Patent Application
20100105054
The terms normal individuals, individuals without Duchenne muscular dystrophy (DMD), control, control group, or controls, patients without DMD, healthy individuals, and normal patients are used synonymously. The terms individuals with DMD, DMD patients, and patients with DMD are used synonymously. Gene expression changes refers to changes in expression of a nucleic acid, also referred to as regulation. Nucleic acid refers to either the sequence that encodes a protein; the nucleic acid can be deoxyribonucleic acid (DNA) or messenger ribonucleic acid RNA (mRNA), or the non-coding sequence that can be either 5′ to the coding sequence (upstream) or 3′ to the coding sequence (downstream). Gene expression changes are not limited to changes in the coding sequence. The nucleic acid sequence can be coding, noncoding (e.g., regulatory sequence such as a promoter), or both coding and noncoding. Evaluation of gene expression encompasses evaluation of mRNA transcription from DNA, and translation into protein, as known by a person having ordinary skill in the art. A described mRNA includes the resultant protein, known by a person having ordinary skill in the art.
DMD is caused by gene mutations in dystrophin, resulting in muscle degeneration associated with chronic inflammation and fibrosis. Strong evidence for immune modulation of muscular dystrophies comes in part from studies of the mdx mouse model of DMD. Treatment with anti-tumor necrosis factor α (anti-TNF-α) protects dystrophic muscle from necrosis. Treatment with cyclosporin A improves function and histology. Expression of perforin, the cytotoxic T-lymphocyte and Natural Killer cell-derived, pro-apoptotic protein is increased in DMD muscle. A null mutation in perforin in the mdx mouse eliminates muscle apoptosis and reduces muscle necrosis. Data suggest an important role for the immune system in the pathogenesis of muscular dystrophy in the mdx mouse.
The immune system also has a role in DMD in humans. Multiple randomized trials have shown improved function and strength in individuals with DMD treated with prednisone (PRED), and with the oxazoline derivative of prednisolone deflazacort (DEFL). It has been argued that since azathioprine, another immunosuppressant, does not improve DMD in humans, the improvements seen with prednisone may not be related to immunosuppression. However, in some immune-mediated disorders such as myasthenia gravis, azathioprine was a less effective immunosuppressant compared to prednisone.
Gene expression profiling of muscle biopsies has been used to examine specific disease-related cell death and cell regeneration pathways in DMD and other muscular dystrophies. Gene expression profiling in muscle also reflects the effects of immune system-modulating treatments; e.g., intravenous immunoglobulin (IV Ig) treatment for inflammatory myopathy changes muscle gene expression.
Clarifying the mechanisms of immune modulation of muscle pathology in muscular dystrophies will provide pathophysiological, diagnostic, and treatment-related implications. Better understanding of these mechanisms at the level of gene expression in muscle is limited by the requirement of an invasive muscle biopsy.
It is known that genetic diseases, such as tuberous sclerosis and neurofibromatosis, and chromosomal disorders, such as Down syndrome, produce disease-specific RNA expression profiles in peripheral whole blood. There are specific RNA expression profiles in peripheral blood following ischemic stroke, likely representing an immune response to injured brain.
The immune response to dying muscle associated with DMD, in addition to the genetic changes associated with DMD, resulted in a specific expression profile in peripheral blood. The demonstrated changes of gene expression, as assessed by measurements of RNA levels from peripheral blood of subjects with DMD compared to controls, on whole genome microarrays, are disclosed.
Individuals with DMD administered the corticosteroids prednisone and deflazacort had improved muscle function and strength. DMD individuals receiving deflazacort had fewer side effects, in particular, less weight gain. Individuals with DMD administered the immunosuppressant azathioprine had no improvement. Benefits from corticosteroid administration may not directly relate or relate entirely to immunosuppression.
Gene expression profiling of muscle tissue has been used to classify muscular dystrophies and examine specific DMD-related cell death and cell regeneration pathways. IV Ig administration for inflammatory myopathy changed gene expression profiles in muscle, evidencing an alteration of the immune response.
Monitoring gene expression changes, e.g., over time, after treatment with an agent, etc. in muscle has, until now, required obtaining a muscle biopsy specimen from the individual. Using peripheral blood as a tissue source in which to monitor gene expression changes eliminated this invasive procedure.
Gene expression changes in blood were used to monitor the effect of steroid administration, resulting in a specific profile of gene expression. Deflazacort and prednisone generated distinct expression profiles. The expression profiles indicated similar mechanisms of action. Deflazacort and prednisone displayed differences in side effects. Overall effects of corticosteroid treatment on mRNA expression in blood of individuals with DMD, and expression specific for deflazacort and specific for prednisone, were identified in a case-controlled whole genome expression microarray study.
One embodiment is a method to evaluate an individual's propensity for DMD. The method comprises determining from blood of a test individual expression of at least one gene from SEQ ID NOS. 1-55; comparing the expression of the gene(s) from the test individual with expression of the same gene(s) from a control individual; evaluating the test individual's propensity for DMD by determining if the gene(s) in the test individual is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to expression of the same gene(s) from the control individual; and ranking the test individual's propensity for DMD based on at least one of (a) the extent that over-expression or under-expression exceeds 1.5, or (b) the number of genes that are over-expressed or under-expressed, where the test individual's propensity for DMD is ranked higher when (a) is farther from 1.5, and (b) is farther from 1 compared to the control individual. In one embodiment the gene is selected from SEQ ID NOS. 1-5, 6-11, 51-55, and/or 46-50. In one embodiment the method further ranks the test individual's propensity higher if at least one of the genes over-expressed or under-expressed is at least one of SEQ ID NOS. 1-5 or SEQ ID NOS. 51-55. In one embodiment, the method ranks the test individual's propensity higher if at least one of the genes over-expressed or under-expressed is SEQ ID NO. 1. In one embodiment, in (a), over-expression or under-expression exceeds 2. In one embodiment, in (a), over-expression or under-expression exceeds 2.5.
One embodiment is a method to diagnose DMD in a test individual. The method comprises determining from blood of a test individual expression of at least one gene selected from SEQ ID NOS. 1-55; comparing expression of the gene(s) from the test individual with expression of the same gene(s) from a control individual; diagnosing DMD by determining if the gene(s) in the test individual is either over-expressed ≧2.5 times or is under-expressed ≧2.5 times compared to expression of the same gene(s) from the control individual; and determining a confidence level for the diagnosis for DMD based on at least one of (a) the extent that over-expression or under-expression exceeds 2.5, or (b) the number of genes that are over-expressed or under-expressed, where the diagnosis for DMD in the test individual has a higher confidence level when (a) is farther from 2.5, and (b) is farther from 1. In one embodiment, the method ranks the confidence level for the diagnosis higher if at least one of the gene(s) over-expressed or under-expressed is at least one of SEQ ID NOS. 1-5 or SEQ ID NOS. 51-55. In one embodiment, the method ranks the confidence level for the diagnosis higher if at least one of the gene(s) over-expressed or under-expressed is defensin.
One embodiment is a method of ranking a gene as a target for ameliorating DMD. The method comprises (a) determining from blood of a test individual with DMD expression of at least one gene selected from SEQ ID NOS. 1-55; (b) comparing the expression of the gene(s) from the test individual with an expression from the same gene(s) from a control individual; and (c) ranking the gene as a target by determining if the at least one gene in the test individual is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to the same gene(s) in the expression profile from the control individual, where the gene is ranked as a better target the farther (c) is from 1.5. In one embodiment, in (c), over-expression or under-expression is ≧2 and the gene is a better target the farther (c) is from 2. In one embodiment, in (c), over-expression or under-expression ≧2.5 and the gene is a better target the farter (c) is from 2.5. In one embodiment, in (a), the gene is at least one of SEQ ID NO. 1-5 or SEQ ID NOS. 51-55. In one embodiment, the method further comprises administering at least one agent specific for at least one of the ranked gene targets, the highest ranked gene, for example, an antibody, an antisense oligonucleotide, etc. In one embodiment, the agent modulates the immune system of the individual with DMD and/or modulates iron utilization of the individual with DMD.
One embodiment is a method of monitoring therapy in an individual with DMD. The method comprises determining from blood of a test individual administered therapy expression of at least one gene selected from SEQ ID NOS. 1-55; comparing expression of the gene(s) from the test individual with expression of the same gene(s) from either (i) a control individual, or (ii) a previous expression result from the test individual; evaluating the test individual's response to therapy by determining if the gene(s) in the test individual is either over-expressed or is under-expressed compared to expression of the same gene(s) from either (i) or (ii), i.e., where the test individual's results after therapy are closer to the control individual's results; and ranking the test individual's response to therapy based on at least one of (a) the extent of over-expression or under-expression, or (b) the number of genes that are over-expressed or under-expressed, where the test individual's response to therapy is ranked higher when at least one of (a) or (b) is closer to either (i) or (ii). In one embodiment, the over-expression or under-expression is ≧1.5. In one embodiment, the over-expression or under-expression is ≧2. In one embodiment, the over-expression or under-expression is ≧2.5.
One embodiment is a kit for diagnosing DMD from peripheral blood. The kit contains at least one primer of a forward oligonucleotide primer and a reverse oligonucleotide primer that anneals to at least one of SEQ ID NO. 1-55; and instructions for using the primer to quantitate SEQ ID NO. 1-55 in peripheral blood. The diagnosis of DMD using the kit is made by determining from blood of a test individual expression of at least one gene selected from SEQ ID NOS. 1-55; comparing expression of the gene(s) from the test individual with expression of the same gene(s) from a control individual; diagnosing DMD by determining if the gene(s) in the test individual is either over-expressed ≧2.5 times or is under-expressed ≧2.5 times compared to expression of the same gene(s) from the control individual; and determining a confidence level for the diagnosis of DMD based on at least one of (a) the extent that over-expression or under-expression exceeds 2.5, or (b) the number of genes that are over-expressed or under-expressed, where the diagnosis for DMD in the test individual has a higher confidence level when (a) is farther from 2.5, and/or (b) is farther from 1. In one embodiment, the primer anneals to defensin. In one embodiment, the primer anneals to T cell leukemia/lymphoma 1a. In one embodiment, the primer is used in the polymerase chain reaction (PCR). In one embodiment, the primer is used in quantitative PCR. In one embodiment, the primer is used in a microarray. In one embodiment, the primer is used in analysis of a Northern blot. Other embodiments are know to a person skilled in the art.
One embodiment is a method of monitoring steroid treatment of DMD. The method comprises determining expression, from blood of a treated individual with DMD administered prednisone and/or deflazacort, at least one gene in Table 4; comparing expression of the gene(s) from the treated individual with an individual with DMD not administered either prednisone or deflazacort or any other steroid; evaluating the treated individual's response to prednisone and/or deflazacort administration by determining if the gene(s) in the treated individual is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to expression of the same gene(s) from the individual with DMD not administered either prednisone or deflazacort or any other steroid; and ranking the treated individual's response to treatment based on at least one of (a) the extent that over-expression or under-expression exceeds 1.5, or (b) the number of genes that are over-expressed or under-expressed, where the treated individual monitored has a greater response to treatment when (a) is farther from 1.5, and (b) is farther from 1. In one embodiment, the ranking is compared to the treated individual's previous expression ranking. In one embodiment, the gene is T cell leukemia/lymphoma 1a. In one embodiment, monitoring determines treatment efficacy. In one embodiment, monitoring determines compliance.
One embodiment is a method of ranking a gene as a target for ameliorating DMD. The method comprises determining from blood of an individual with DMD administered prednisone and/or deflazacort expression of at least one gene in Table 4; (b) comparing expression of the gene(s) from (a) with expression of the same gene(s) from an individual with DMD not administered either prednisone or deflazacort; and (c) ranking the gene(s) as a target for ameliorating DMD by determining if the gene(s) in (a) is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to the same gene(s) in the individual with DMD not administered prednisone or deflazacort, where the gene is a better target the farther (c) is from 1.5. In one embodiment, in (c), over-expression or under-expression is ≧2 and the gene(s) is a better target the farther (c) is from 2. In one embodiment, in (c) over-expression or under-expression is ≧2.5 and the gene is a better target the farther (c) is from 2.5.
One embodiment is a method of determining amelioration of prednisone side effects in an individual with DMD. The method comprises ranking a gene as a target for ameliorating prednisone side effects by (a) determining from blood of an individual with DMD administered prednisone expression of at least one gene selected from Table 5; (b) comparing the expression in (a) with expression of the same gene(s) from an individual with DMD administered deflazacort, and (c) ranking the gene as a target by determining if the gene in (a) is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to the same gene in expression from (b), where the gene is a better target the farther (c) is from 1.5. In one embodiment, at least one agent specific for the gene is then prescribed. In one embodiment, at least one of the determined targets is responsible for the side effects caused by prednisone compared to deflazacort.
One embodiment is a method of determining efficacy of treatment of DM. The method comprises determining, from blood of a treated individual with DMD administered a steroid, expression of at least one gene in Table 4; comparing expression of the gene(s) from the treated individual with expression of the same gene(s) in an individual with DMD not treated with a steroid; evaluating treatment efficacy by determining if the gene(s) in the treated individual is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to expression of the same gene(s) from the individual with DMD not treated with a steroid; and determining treatment efficacy based on at least one of (a) the extent that over-expression or under-expression exceeds 1.5, or (b) the number of genes that are over-expressed or under-expressed, where the treatment is more efficacious when (a) is farther from 1.5, and/or (b) is farther from 1. In one embodiment, the gene encodes T cell leukemia/lymphoma 1a. In one embodiment, the gene encodes defensin.
One embodiment is a kit for monitoring steroid treatment from peripheral blood in an individual with DMD. The kit contains at least one primer of a forward oligonucleotide primer and a reverse oligonucleotide primer that anneals to at least one of the genes in Table 4; and instructions for using the primer to quantitate the gene(s) in Table 4 in peripheral blood. Monitoring is by determining expression, from blood of a treated individual with DMD administered prednisone and/or deflazacort, at least one gene in Table 4; comparing expression of the gene(s) from the treated individual with an individual with DMD not administered a steroid; evaluating the treated individual's response to prednisone and/or deflazacort administration by determining if the gene(s) in the treated individual is either over-expressed ≧1.5 times or is under-expressed ≧1.5 times compared to expression of the same gene(s) from the individual with DMD nor administered prednisone and/or deflazacort; and ranking the treated individual's response to treatment based on at least one of (a) the extent that over-expression or under-expression exceeds 1.5, or (b) the number of genes that are over-expressed or under-expressed, where the treated individual has greater response to treatment when (a) is farther from 1.5, and (b) is farther from 1. In one embodiment, the primer anneals to the gene encoding defensin. In one embodiment, the primer anneals to the gene encoding T cell leukemia/lymphoma 1a. In one embodiment, the primer is used in PCR. In one embodiment, the primer is used in qPCR. In one embodiment, the primer is used in a microarray. In one embodiment, the primer is used in analysis of a Northern blot. Other embodiments are known to a person skilled in the art.
In one embodiment, a gene expression profile characteristic of DMD comprised at least one gene in SEQ. ID NOS. 1-55.
In one embodiment, a gene expression profile characteristic of DMD comprised at least one of nucleic acids 1-59, shown in Table 1.
In one embodiment, a gene expression profile characteristic of DMD comprised at least one of nucleic acids 1-191, shown in Table 2.
In one embodiment, a gene expression profile characteristic of DMD comprised at least one of nucleic acids 1-1467, shown in Table 3.
In addition to listing the nucleic acids, Tables 1, 2, and 3 disclose the relative extent of regulation and whether regulation is by over-expression or by under-expression.
In one embodiment, DMD was diagnosed by evaluating gene expression or gene expression profiles. In one embodiment, DMD was diagnosed by determining expression of at least one gene selected from SEQ. ID NOS. 1-55 in peripheral blood of the individual. Expression of the selected gene(s) was then compared to expression of that same gene(s) in an individual that did not have DMD. The individual was diagnosed for DMD by determining if the gene in the individual was either over-expressed ≧2.5 times or was under-expressed ≧2.5 times, compared to expression of the same gene in the individual without DMD. Confidence in the diagnosis, described as a confidence level of the diagnosis, was determined by evaluating the extent that over-expression or under-expression exceeded 2.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the diagnosis for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 2.5, (b) whether the gene was from SEQ ID NO. 1-55 and/or (c) the greater the number of genes that were over-expressed or under-expressed. In one embodiment, the gene expression profile comprises defensin (SEQ ID NO. 1).
In one embodiment, DMD was diagnosed by determining an expression profile of at least one gene selected from Nucleic Acid Nos. 1-191 in peripheral blood of the individual. The expression profile of the selected gene(s) was then compared to the expression profile of that same gene in an individual that did not have DMD. The individual was diagnosed for DMD by determining if the gene in the individual was either over-expressed ≧2.0 times or was under-expressed ≧2.0 times, compared to the same gene in the expression profile in the individual without DMD. A confidence level of the diagnosis was determined by evaluating the extent that over-expression or under-expression exceeded 2.0, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the diagnosis for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 2.0, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed.
In one embodiment, DMD was diagnosed by determining an expression profile of at least one gene selected from Nucleic Acid Nos. 1-1467 in peripheral blood of the individual. The expression profile of the selected gene(s) was then compared to the expression profile of that same gene in an individual that did not have DMD. The individual was diagnosed for DMD by determining if the gene in the individual was either over-expressed ≧1.5 times or was under-expressed ≧1.5 times, compared to the same gene in the expression profile in the individual without DMD. A confidence level of the diagnosis was determined by evaluating the extent that over-expression or under-expression exceeded 1.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the diagnosis for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 1.5, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed.
In one embodiment, a gene expression profile from peripheral blood showing increased defensin SEQ ID NO.: 1 in an individual, compared to a gene expression profile for defensin mRNA in a normal individual, was used to diagnose DMD. In one embodiment, defensin mRNA was expressed greater than 2.5 fold higher than defensin mRNA was expressed in a normal individual.
In one embodiment, a gene expression profile from peripheral blood showing decreased utrophin Nucleic Acid No. 1378 in an individual, compared to a gene expression profile for utrophin mRNA in a normal individual, was used to diagnose DMD. In one embodiment, utrophin mRNA was expressed less than 1.5 fold lower than utrophin mRNA was expressed in a normal individual.
In one embodiment, gene expression profiles in an individual were used to evaluate DMD disease propensity, disease severity, disease subtype, therapy efficacy, therapy compliance, an individual's response to therapy, etc. In one embodiment, DMD was evaluated by determining an expression profile of at least one gene selected from SEQ. ID NOS. 1-55 in peripheral blood of the individual. In one embodiment, DMD was evaluated by determining an expression profile of at least one gene selected from Nucleic Acid Nos. 1-191 in peripheral blood of the individual. In one embodiment, DMD was evaluated by determining an expression profile of at least one gene selected from Nucleic Acid Nos. 1-1467 in peripheral blood of the individual. The expression profile of the selected gene(s) was then compared to the expression profile of the same gene(s) in an individual that did not have DMD.
The individual's disease propensity, disease severity, and/or disease subtype for DMD was evaluated by determining if the gene in the individual was either over-expressed ≧1.5 times or was under-expressed ≧1.5 times, compared to the same gene expressed in the individual without DMD. Propensity was evaluated by determining the extent that over-expression or under-expression exceeded 1.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The individual's propensity for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 1.5, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed. In one embodiment, the gene encoded defensin (SEQ ID NO. 1). In one embodiment, propensity for DMD was distinguished from diagnosis of DMD, where propensity for DMD was determined in an asymptomatic and/or undiagnosed individual. In one embodiment, disease severity was determined in a symptomatic individual. In one embodiment, disease subtype was determined in an asymptomatic individual or in a symptomatic individual. For example, an individual's propensity for a particular DMD subtype may be determined, or an individual may be diagnosed as having a particular DMD subtype.
The individual's therapy efficacy, therapy compliance, and/or response to therapy may be termed therapy monitoring. The individual's therapy for DMD was monitored by determining if the gene in the individual was either over-expressed ≧1.5 times or was under-expressed ≧1.5 times, compared to the same gene(s) expressed in the individual without DMD. Therapy was monitored by determining the extent that over-expression or under-expression exceeded 1.5, the identity of the gene(s) over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The efficacy of therapy, compliance with therapy, and or response to therapy for DMD was lower, based on: (a) the farther the over-expression or under-expression was from 1.5, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed. In one embodiment, the gene encoded defensin.
In one embodiment, at least one steroid was administered as therapy for individuals with DMD and gene expression was compared to gene expression in individuals with DMD not administered any steroids. In one embodiment, corticosteroids were administered as therapy for individuals with DMD. In one embodiment, prednisone was administered as therapy for individuals with DMD. In one embodiment, deflazacort was administered as therapy for individuals with DMD. In one embodiment, deflazacort and prednisone were administered as therapy for individuals with DMD. In one embodiment, the efficacy or compliance to steroid, corticosteroid, prednisone and/or deflazacort treatment for DMD was evaluated by determining expression of at least one gene selected from nucleic acids 1-524 (Table 4) in peripheral blood of the individual. Nucleic acids 1-524 represented those nucleic acids that were either over-expressed ≧1.5 times or were under-expressed ≧1.5 times, compared to the same nucleic acids expressed in an individual with DMD not administered any steroids.
In one embodiment, the efficacy or compliance to prednisone and/or deflazacort treatment for DMD was evaluated by determining expression of at least one gene selected from nucleic acids 1-508 (Table 5) in peripheral blood of the individual. Nucleic acids 1-508 represented those nucleic acids expressed in an individual with DMD and administered prednisone that were either over-expressed ≧1.5 times or under-expressed ≧1.5 times, compared to the same nucleic acids expressed in an individual with DMD and administered deflazacort. In one embodiment, the efficacy or compliance to prednisone treatment for DMD was evaluated by determining if the gene in the individual was either over-expressed 1.5 times or was under-expressed ≧1.5 times, compared to the same gene in the expression profile in an individual with DMD and treated with deflazacort. In one embodiment, the efficacy of or compliance with deflazacort administration for DMD was evaluated by determining if the gene in the individual was either over-expressed ≧1.5 times or was under-expressed ≧1.5 times, compared to the same gene in the expression profile in an individual with DMD and administered prednisone.
In one embodiment, a therapeutic target for DMD treatment was determined by evaluating gene expression. In one embodiment, a therapeutic target for DMD treatment was determined by determining expression of at least one gene selected from SEQ. ID NOS. 1-55 in peripheral blood of the individual with DMD. Expression of the selected gene(s) was then compared to expression of that same gene in an individual that did not have DMD. The gene(s) was determined to be a therapeutic target for DMD treatment by determining if the gene in the individual was either over-expressed ≧2.5 times or was under-expressed ≧2.5 times, compared to the same gene expressed in the individual without DMD. A confidence level of the therapeutic target was determined by evaluating the extent that over-expression or under-expression exceeded 2.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the therapeutic target for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 2.5, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed. In one embodiment, the gene expression profile comprises defensin.
In one embodiment, a therapeutic target for DMD treatment was determined by determining expression of at least one gene selected from nucleic acids 1-1467 in peripheral blood of the individual with DMD. Expression of the selected gene(s) was then compared to expression of the same gene(s) in an individual that did not have DMD. The gene(s) was determined to be a therapeutic target for DMD treatment by determining if the gene(s) in the individual was either over-expressed ≧1.5 times or was under-expressed ≧1.5 times, compared to the same gene(s) in expressed in the individual without DMD. A confidence level of the therapeutic target was determined by evaluating the extent that over-expression or under-expression exceeded 1.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the therapeutic target for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 1.5, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed.
In one embodiment, a therapeutic target for DMD treatment was determined by determining expression of at least one gene selected from nucleic acids 1-191 in peripheral blood of the individual with DMD. Expression of the selected gene(s) was then compared to expression of that same gene in an individual that did not have DMD. The gene(s) was determined to be a therapeutic target for DMD by determining if the gene(s) in the individual was either over-expressed ≧2.0 times or was under-expressed ≧2.0 times, compared to the same gene(s) expressed in the individual without DMD. A confidence level of the therapeutic target was determined by evaluating the extent that over-expression or under-expression exceeded 2.0, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The confidence level of the therapeutic target for DMD was higher, based on: (a) the farther the over-expression or under-expression was from 2.0, (b) whether the gene was from SEQ ID NO. 1-55, and/or (c) the greater the number of genes that were over-expressed or under-expressed.
In one embodiment, an agent's effect on the pathophysiology of DMD was evaluated. At least one cell, referred to as the test cell, was exposed to the agent, and expression of at least one gene selected from SEQ ID NOS. 1-55 in the cell(s) was compared to expression of the same gene(s) selected from SEQ ID NOS. 1-55 in a cell of an individual without DMD, referred to as the control cell. The contribution of the agent to the pathophysiology of DMD was evaluated by determining if the gene(s) in the test cell was either over-expressed ≧1.5 times, or was under-expressed ≧1.5 times, compared to the same gene(s) expressed in the control cell. The agent's contribution to the pathophysiology of DMD was evaluated by determining the extent that over-expression or under-expression exceeded 1.5, the identity of the gene over-expressed or under-expressed, and/or the number of genes that were over-expressed or under-expressed. The agent contributed more to the pathophysiology of DMD based on at least one of the farther the over-expression or under-expression was from 1.5, the gene was from SEQ ID NO. 1-55, and/or the greater the number of genes that were over-expressed or under-expressed. In one embodiment, expression of at least one gene selected from nucleic acids 1-191 was compared. In one embodiment, expression of at least one gene selected from nucleic acids 1-1467 was compared. In one embodiment, the cell was a component of peripheral blood. In one embodiment, based on the extent that the agent contributed to DMD, agents that antagonized the action of the agent were predicted or used to treat DMD.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Except where specifically indicated, gene expression in all individuals and all gene expression data were evaluated from peripheral blood.
Individuals with DMD, some of which had been administered prednisone and/or deflazacort, compared to controls, had significant differences in gene expression, which was the first demonstration of gene expression changes in peripheral blood on a whole genome level.
A total of 10,763 genes were significantly regulated in peripheral blood of these individuals with DMD, compared to individuals without DMD. Of the 10,763 genes that were significantly regulated, with regulation including genes that were either up- or down-regulated, the data were:
1467 genes had a fold change >|1.5|;
191 genes had a fold change >|2.0|; and
59 genes had a fold change >|2.5|, with fold changes indicated as absolute values.
The regulated genes in individuals with DMD were expressed mainly in neutrophils, monocytes, B cells, and CD4+ T cells. This mirrored to some extent the muscle infiltration of monocytes/macrophages and T-cells in individuals with DMD. Different genes were activated in the blood, compared to muscle, in individuals with DMD, though there were similarities in the functional pathways.
The gene encoding utrophin (nucleic acid 1378) was regulated in both blood and muscle of individuals with DMD. The utrophin protein is highly homologous to dystrophin (80% homology). Dystrophin is deficient in individuals with DMD. In blood, utrophin was down-regulated two-fold in individuals with DMD compared to controls. In muscle, utrophin is up regulated in individuals with DMD compared to controls, presumably as a partial compensation for the dystrophin deficiency. The two-fold down-regulation of utrophin in blood presumably relates to a change in immune function of the white cells in individuals with DMD, rather than as a consequence of the dystrophin deficiency in muscle.
In individuals with DMD, some genes that are normally expressed in muscle were regulated in blood, but were not regulated in muscle. Some of these genes regulated in blood, but not muscle, of individuals with DMD include myoferlin, sarcolemma associated protein (SLMAP), myocyte enhancer factor 2C (MEF2C), myotubularin related protein 1 (MTMR1), and phosphodiesterase 4D interacting protein (PDE 4DIP) (nucleic acids 1405, 1465, 1232, 1423, 1464, and 80, respectively).
Myoferlin was down-regulated two fold in blood of individuals with DMD but, although present, was not regulated in muscle of individuals with DMD. Myoferlin has an unknown function, but is highly homologous to dysferlin, which is mutated in some types of limb-girdle muscular dystrophy. Myoferlin protein levels do not appear to be dysregulated in subjects with dysferlinopathy.
Sarcolemma associated protein (SLMAP) was down-regulated in blood of individuals with DMD but, although present, was not regulated in muscle of individuals with DMD. The coiled-coil, tail-anchored SLMAP protein self-organizes in the cardiomyocyte and may be involved in the excitation coupling apparatus.
Myocyte enhancer factor 2C (MEF2C), myotubularin related protein 1 (MTMR1), and phosphodiesterase 4D interacting protein (PDE 4DIP, myomegalin) were regulated in blood of individuals with DMD but, although present, were not regulated in muscle of individuals with DMD. MEF2 genes control skeletal muscle development and MEF2C mediates the activation of induced cell death (AICD) of macrophages. Mutations of myotubularin-like proteins lead to myotubular myopathy and a form of Charcot-Marie-Tooth peripheral neuropathy. The myotubularin family of PI 3-phosphatases is a key regulator of two phosphoinositols that regulate traffic within the endosomal-lysosomal pathway. Myomegalin functions as an anchor to localize components of the cAMP-dependent pathway to the Golgi/centrosomal region of the cell.
Regulation of genes encoding myoferlin, sarcolemma associated protein (SLMAP), myocyte enhancer factor 2C (MEF2C), myotubularin related protein 1 (MTMR1), and phosphodiesterase 4D interacting protein (PDE 4DIP) point to pathways that are activated in white blood cells in individuals with DMD, and to pathways that are utilized within normal muscle cells but are not activated in the diseased muscle in individuals of DMD. These genes likely participated in functions of immune cells that were activated in response to diseased DMD muscle cells.
There was significant overlap in the signaling pathways that were activated in blood and muscle of individuals with DMD, including the leukocyte trans-endothelial migration pathway (
Amyloid precursor protein, cellular prion protein, and caspase 1 were up-regulated in individuals with DMD. Caspase-1, which cleaves interleukin 1 produced by white blood cells, could be involved in white blood cell apoptosis or regulating pro-inflammatory IL-1 levels in white blood cells. Caspase-1 up regulation may contribute to changes of oxidative stress and possible role of apoptosis in cell death in DMD muscle.
To identify pathways with the most differentially expressed genes, gene lists were searched against the KEGG database of human biochemical pathways using the KEGG pathway functional annotation tool in DAVID Bioinformatics Resources: http://david.abcc.ncifcrf.gov/. The top biochemical pathways identified by KEGG in blood of individuals with DMD included leukocyte transendothelial migration and antigen processing pathways; both are closely related to immune function. The monocytes/macrophages and T cell infiltration in muscle of individuals with DMD, and with dysferlin myopathy, is a well-recognized inflammatory response. Most individuals with DMD express a highly conserved peptide in the hypervariable domain of the T-cell receptor in cytotoxic lymphocytes, suggesting a specific immune response to a common antigen.
Immune cells, such as macrophages and T cells, promote the pathology of dystrophic muscle. Prednisone and prednisolone improve muscle strength in dystrophin deficient mdx mice, reduce muscle degeneration in dystrophin deficient Caenorhabditis elegans (C. elegans), decrease CD-11 inflammatory cells in mdx muscle, and improve function in individuals with DMD. Prednisone thus appears to improve function, at least in part, by decreasing inflammation and improving survival of diseased muscle fibers. The discloses data support a significant immune response in blood of individuals with DMD that involves several cell types including neutrophils, monocytes/macrophages, B cells, and T-cells. An agent that targeted one or more individually regulated genes would more specifically modulate the immune system and improve function in individuals with DMD. The agent may up-regulate gene expression. The agent may down-regulate gene expression. Examples of agents include, but are not limited to, an antisense oligonucleotide, an inhibitory RNA (RNAi), an agonist of the resultant protein, an antagonist of the resultant protein, an expression vector of the regulated gene, an antibody, etc. In one embodiment, an anti-human monoclonal antibody may be administered. In one embodiment, an antisense oligonucleotide for defensin may be administered. In one embodiment, a define RNAi may be administered.
The 1467 genes that had a fold change >|1.5| (Table 3, nucleic acids 1-1467), the 191 genes that had a fold change >|2.0| (Table 2, nucleic acids 1-78 and 1355-1467), and the 59 genes that had a fold change >|2.5| (Table 1, nucleic acids 1-45 and 1454-1467, and SEQ ID NOS. 1-55) were evaluated by cluster analysis to determine capability to separate individuals with DMD compared to control individuals. The results are shown in
An RNA expression profile of individuals with DMD compared to control individuals was generated. The immune response to necrosis/apoptosis of dystrophic muscle likely accounts, at least in part, for the changes of RNA expression. RNA changes might provide an assay for monitoring targeted therapies for DMD and other neuromuscular disorders.
Gene expression and/or gene expression profiles were identified in individuals with DMD that were administered the agents deflazacort, prednisone, or deflazacort and prednisone. Gene expression changes common to administration of either deflazacort or prednisone related to the efficacy of deflazacort therapy, prednisone therapy, or deflazacort and prednisone therapy in individuals with DMD. In addition, there were gene expression changes that were specific for deflazacort or prednisone that might relate to deflazacort- or prednisone-specific side effects or therapeutic actions.
When gene expression in individuals with DMD not receiving deflazacort or prednisone, were compared to gene expression in individuals with DMD receiving deflazacort, prednisone, or deflazacort and prednisone, expression of 524 genes was significantly different. The results are shown in Table 4,
Using these data in a prediction analysis of microarray (PAM) to cross-validate probabilities, 59 probes representing 50 annotated genes were identified that optimally distinguished patients with DMD receiving deflazacort, prednisone, or deflazacort and prednisone, from patients with DMD not receiving deflazacort, prednisone, or deflazacort and prednisone. Table 6. Ten-fold leave-one-out cross-validation demonstrated that these probes correctly classified 29 of 34 individuals (85.2%) administered deflazacort, prednisone, or deflazacort and prednisone.
The individuals with DMD administered deflazacort or prednisone, compared to individuals with DMD not administered deflazacort or prednisone, demonstrated enhanced defense responses and up-regulation of genes associated with primary and secondary granules in granulocytes (neutrophils), iron trafficking, and chondroitin sulfate synthesis. The individuals with DMD administered deflazacort or prednisone, compared to individuals with DMD not administered deflazacort or prednisone, demonstrated increased expression levels of lactotransferrin and lipocalin, genes involved in iron and heme homeostasis. Lactotransferrin, an iron binding protein found in secondary granules of neutrophils, is important in the innate immune response against infections, and appears to be critically important in the oxidative burst during infections. These results confirmed a previous study that demonstrated that corticosteroid administration to normal individuals increased lactotransferrin blood levels. Lipocalin 2 is important in removing or preventing iron from entering siderophore bound iron in bacteria. Lipocalin 2 also binds and carries iron in neutrophils, and plays a role in resistance against tissue injury. Both lactotransferrin and lipocalin 2 traffic iron to late endosomes where acidification and reduction processes release iron stores. Steroids have previously been reported to induce lipocalin both in vitro and in vivo.
The individuals with DMD administered deflazacort or prednisone, compared to individuals with DMD not administered deflazacort or prednisone, demonstrated up-regulated expression levels of haptoglobin and CD163 nucleic acids (nucleic acids 1641 and 1526, respectively). Haptoglobin, made predominantly in the liver, binds hemoglobin and other heme proteins. It is specifically taken up by the CD163 receptor into macrophages, thus sequestering heme and iron and preventing oxidative damage. The up-regulation of haptoglobin could serve to bind and sequester heme proteins released from injured or dying muscle fibers in DMD and other neuromuscular diseases. Corticosteroids induce haptoglobin protein production in blood of mammals and in cultured cells. The results showing increased haptoglobin expression in response to corticosteroids in humans confirmed these previous studies.
The disclosed results are consistent with a previous human study showing that corticosteroids also induced CD163 protein on the surface of peripheral blood monocytes. That study suggested that corticosteroids facilitated CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induced CD163 and heme oxygenase-1 (HO-1) synthesis. The HO-1 metabolism of heme from heme-containing proteins and sequestration of the released iron could account for some of the anti-inflammatory actions of corticosteroids.
Several of the disclosed up-regulated genes, including lactotransferrin, cathepsin G, elastase, and azurocidin, directly bound ceruloplasmin. Ceruloplasmin is an iron binding protein and an endogenous antioxidant that oxidizes Fe2+ to Fe3+, mitigating the oxidant effect of iron. While some of the disclosed corticosteroid up-regulated genes were reported previously in other systems, the disclosed results demonstrated the coordinated induction of iron and heme regulatory genes in human leukocytes in response to corticosteroids. This is particularly interesting in light of therapeutic effects of iron deprivation demonstrated in the mdx mouse.
The individuals with DMD administered deflazacort or prednisone, compared to individuals with DMD not administered deflazacort or prednisone, demonstrated up-regulated expression levels of genes in the chondroitin sulfate biosynthesis pathway. Biglycan and decorin, chondroitin/dermatan sulfate proteoglycans found in muscle extracellular matrix, have been shown to increase in muscle of individuals with DMD. Biglycan and decorin may act as regulators of fibrosis through inhibition of TGF-B1 profibrotic cytokine activity. However, reduced proteoglycan sulfonation in and at the basal lamina has been identified in cultured DMD muscle cells. The disclosed up-regulation of chondroitin β-1,4 N-acetylgalactosaminyltransferase (ChGn, GALNACT-2) in response to corticosteroids might normalize proteoglycan sulfonation and enhance anti-fibrotic effects of biglycan and decorin by contributing to initiation and elongation in chondroitin sulfate biosynthesis.
The individuals with DMD administered deflazacort or prednisone, compared to individuals with DMD not administered deflazacort or prednisone, demonstrated up-regulated expression levels of FKBP5. The up-regulation of FKBP5 in response to corticosteroid use has been consistently demonstrated, including in the mdx mouse, and has been associated with loss of corticosteroid efficacy. The FKBP5 protein inhibits calcineurin, which controls and regulates slow-twitch oxidative (type 1) muscle fibers. Manual curation comparing expression changes in the mdx mouse identified only one regulated probe in addition to FKBP5; this was KFL10. KFL10, up-regulated following six weeks of prednisone administration in the mdx mouse, was also up-regulated in individuals administered prednisone, deflazacort, or both, compared with individuals DMD not administered any agent. KLF10 is a TGF-β-inducible early gene that acts as a transcription factor regulator binding to GC-rich Sp1-like sequences. Thus it is possible that although downstream expression changes differ between muscle and blood in response to steroid administration in individuals with DMD, these cascades are initiated by similar mechanisms.
None of the genes having deflazacort- or prednisone-altered expression in individuals with DMD had glucocorticoid (corticosteroid) response elements (GRE) significantly over- or under-represented in transcription factor binding motifs. The most significantly over-represented transcription factor binding motif family was the myocyte-specific enhancer factor-2 (MEF2) family. The proteins in the MEF2 family bind to the MEF2 DNA sequence in regulatory regions of most muscle-specific genes. Other over-represented transcription factor motifs included CEBPA, Oct 6 and Brn 2. CEBPA (CCAAT enhancer binding proteins) are transcription factors involved in specification of myeloid lineages (i.e., all white blood cell types except lymphocytes and monocytes) from stem cells and up-regulation of expression for primary and secondary granule proteins. The shift to a predominantly myeloid lineage immune response, from lymphocyte defense responses, in response to corticosteroids is further supported by under representation of binding sites for Ikaros transcription factors, transcription factors regulating lymphocyte differentiation. The transcription factors Oct6 and Brn2 interact to promote the transition from promyelinating to myelinating Schwann cells. Since the short form of dystrophin is found in peripheral myelin, over-representation of Oct6 and Brn2 transcription factor binding sites is intriguing Enhanced proteolytic activity by matrix metalloproteinase 9 (MMP-9) in mdx mouse, suggested to arise from increased inflammation and subsequent macrophage activation, might potentiate myelin degeneration in dystrophin-deficient Schwann cells. Thus the over representation of the Oct6 and Bm2 transcription factors suggest a possible remyelination response due to corticosteroid administration in individuals with DMD.
The individuals with DMD administered deflazacort, compared to the individuals with DMD administered prednisone, demonstrated 508 genes nucleic acids 1-508 that were significantly different. The results are shown in Table 5,
In the individuals with DMD administered deflazacort, expression of retinoic acid receptor a (RARa) was significantly up-regulated compared to individuals administered prednisone. Retinoic acid inhibits adipocyte differentiation through RARa, and increased expression of RARa has been associated with reduced obesity levels.
In the individuals with DMD administered prednisone, expression of a number of genes related to lipid metabolism, particularly in adipose tissue, was up-regulated compared to individuals administered deflazacort. For example, Cas-Br-M (murine) ecotropic retroviral transforming sequence (CBL) is involved in adipose formation and energy homeostasis. The nicotinic acid-coupled G protein-coupled receptor 109B (GPR109B), and the G-protein-coupled receptor 43, a short-chain fatty acid receptor, each inhibits niacin-mediated lipolysis in adipose tissue. Nuclear receptor co-activator 1 (NCOA1), a co-activator required for transcriptional activity of the steroid receptor superfamily, is involved in energy metabolism. The interleukin 1 and interleukin 6 systems are both associated with obesity. Individuals with DMD administered prednisone had up-regulated interleukin 1 beta, interleukin 1 receptor antagonist, and interleukin 6 receptor. These results suggested a mechanism by which weight gain, an important side effect of prednisone administration, might be ameliorated, as well as a mechanism by which weight gain might be inhibited with deflazacort, for example, by administering agents that perturb regulation. There were fewer changes in gene expression in individuals that had been administered deflazacort compared to individuals that been administered prednisone. The gene expression profile of individuals administered deflazacort is more similar to the gene expression profile of individuals with DMD not administered prednisone or deflazacort, that to the gene expression profile of individuals with DMD administered prednisone.
Corticosteroid administration fostered myeloid maturation, may suppress fibrosis, and appeared to up-regulate genes for primary and secondary granule proteins and subsequent iron trafficking functions in individuals with DMD. The disclosed results indicated pathways involved in weight gain and metabolism that are up-regulated in individuals with DMD administered prednisone but not in individuals with DMD administered deflazacort. The association of these pathways with corticosteroid administration points to mechanisms of corticosteroid actions in DMD that might provide specific treatment targets for other agents without side effects of corticosteroids.
Gene expression from 34 males with DMD, ranging from 3 years to 20 years with a mean age of 9 years±3 years, was compared to gene expression in 21 control males matched for age. Table 8. Individuals with DMD were evaluated and had been diagnosed by a single board-certified pediatric neurologist. The diagnosis of DMD was based in part upon clinical history, examination findings, and selected muscle biopsies and was confirmed by molecular diagnostic testing.
Of the 34 individuals with DMD, 14 were administered either prednisone (PRED, n=6) or deflazacort, an oxazoline derivative of prednisolone (DEFL; n=8) at the time blood samples for gene expression evaluation were obtained. The mean duration of administration of either prednisone or deflazacort for these 14 individuals was 43.9 months. Of the 34 individuals with DMD, 20 individuals had not been administered either prednisone or deflazacort at the time blood samples for gene expression evaluation were obtained and details of clinical and genetic testing are available upon request.
There was a non-significant age difference (t-test, p>0.20) between the control individuals and the individuals administered either prednisone or deflazacort. One control individual and 3 individuals with DMD were not Caucasian. An unpaired t-test was performed when comparing results among all of the individuals (both control individuals and individuals with DMD), and a paired t-test was performed when comparing equal numbers of age-matched, male, Caucasian control individuals and individuals with DMD, as subsequently shown.
The control individuals were recruited from local schools with similar demographics to those from which individuals with DMD attended, and blood was obtained and processed using identical methods for both groups.
Sample Processing and Array Hybridization
Fifteen ml of blood was collected from each individual into 6 PAXgene® Vacutainer® tubes (PreAnalytiX, Germany) by antecubital fossa venipuncture. PAXgene® tubes contain a proprietary reagent that immediately stabilizes RNA, thus reducing RNA degradation and minimizing gene expression changes following phlebotomy. The tubes were then inverted 20 times, then remained undisturbed for 2 h at room temperature (about 19° C. to about 22° C.), and then stored at −70° C. until processed.
Total RNA was isolated using the Paxgene® blood RNA kit (PreAnalytiX, Germany) according to the manufacturers instructions. The RNA isolated is from all cells in whole blood. Typical yields of total RNA isolated from about 7.5 ml of human whole blood were between 10 μg RNA and 40 μg RNA. RNA quality and purity was analyzed by spectrophotometry using the Nanodrop ND-1000, and RNA integrity was analyzed using the Agilent 2100 Bioanalyzer. A260/A280 of purified RNA exceeded 2.0, and the 28S/18S rRNA ratios were equal to or exceeded 1.8.
Sample labeling, hybridization to chips, and image scanning were performed using standard Affymetrix protocols. For each whole blood sample from control individuals and individuals with DMD, total RNA was labeled using the One-Cycle Target Labeling protocol and hybridized to the arrays. Gene expression was assessed on the Human U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara Calif.), which consisted of a single oligonucleotide microarray that surveyed over 54,000 probe sets, or greater than 30,000 possible human genes. Double-stranded cDNA synthesis, biotin-labeled cRNA synthesis, and cRNA fragmentation were performed according to the Affymetrix GeneChip Eukaryotic Expression Analysis protocol. The labeled, fragmented cRNA (15 μg) was hybridized at 45° C. overnight. After hybridization, the array was washed and stained. The arrays were scanned with an argon-ion laser at λ=570 nm, with a resolution of 3 μm/pixel (Affymetrix, Inc.).
Probe-Level Data Analysis
After scanning the array, the raw probe level values for each probe were saved in Affymetrix.cel files. The probe level values were imported into Genespring 7.2 (Agilent Technologies, Palo Alto Calif.), and then processed using GC-RMA, followed by a three-step normalization (data transformation, per chip normalization, and per gene normalization) (Genespring 2004).
Statistical Analyses
Statistical analyses, including t-test analyses, unsupervised hierarchical clustering, and principal component analyses were conducted using Genespring 7.2 software (GENESPRING® 7, Silicon Genetics, Redwood City Calif.). Significance was assessed using the criteria at least 1.5-fold difference in expression between groups and statistically significantly different level of expression (parametric t-tests, p≦0.05). Corrections for multiple comparisons used a Benjamini-Hochberg False Discovery Rate (FDR) of 0.05, disclosed in Tang et al., J Cereb Blood Flow Metab 26 (2006) 1089, which is expressly incorporated by reference herein in its entirety. This value was chosen because no more than 5% of the genes should represent false positives.
Results of the t-tests were reported without a fold change cutoff. Genes for which the fold change in the average expression values in individuals with DMD, compared to control individuals, were >1.5 (least stringent), >2.0 (moderately stringent), or >2.5 (very stringent). The gene lists were used for cluster analyses to determine how well the genes separated individuals with DMD from control individuals. The increasing degrees of stringency provided manageable numbers of genes; primary data are available upon request. Previous blood studies evaluating gene expression in neurological diseases had often used 1.5 fold changes. Approaches that combined significance value measures and magnitude of change measures provided good power and prediction, as shown for Significance Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM).
Prediction analysis of microarray (PAM) software, which employs the “nearest shrunken centroids” method, was used to identify a minimum set of signature genes that distinguished individuals with DMD from control individuals. The accuracies of the classifiers were tested using a tenfold cross-validation.
Assessment of Possible Biological Significance of Identified Genes
Gene lists were searched against the KEGG database of human biochemical pathways (using the KEGG pathway functional annotation tool in DAVID Bioinformatics Resources: http://david.abcc.ncifcrf.gov/) to identify pathways with the most differentially expressed genes. A probability p value for the numbers of genes expressed in a given pathway was used to select the most regulated pathways in blood of individuals with DMD.
The primary gene expression data published for muscle from individuals with DMD, compared to control individuals, was re-analyzed using GCRMA. The regulated genes in muscle of individuals with DMD subjects were compared to the regulated genes in peripheral blood of individuals with DMD. The most prominent pathways regulated in blood were compared to those regulated in muscle for individuals with DMD (
When gene expression was evaluated from the 34 individuals with DMD, compared to the 21 control individuals, 10,763 genes were found to be significantly regulated (unpaired t-test and a Benjamini-Hochberg False discovery rate (FDR), (p≦0.05), no cutoff for fold change, data not shown). Of the 10,763 genes that were significantly regulated, 59 genes showed a fold change >|2.5| (Table 1), 191 genes showed a fold change >|2.0| (Table 2), and 1467 genes showed a fold change of >|1.5| (Table 3).
A separate analysis was performed on equal sized groups matched for gender (male), race (Caucasian), and age (3 years to 20 years). A paired t-test for individuals with DMD (n=20) compared to control individuals (n=20) resulted in lists of genes that overlapped the above gene lists, using a FDR of <0.05 and the fold changes indicated above (data not shown).
Using the data in Table 1 (which lists the 59 genes differentially regulated in individuals with DMD compared to control individuals using an unpaired t-test, FDR <0.05, fold change >2.5), a cluster analysis separated individuals with DMD from control individuals. The results are shown in
Control individuals formed a single group with a block of genes (lower left) that were generally expressed at higher levels in control individuals, compared to individuals with DMD (lower right).
Based upon the patterns of gene expression for individuals with DMD, different subgroups of genes were discerned. These subgroups appeared to further distinguish among individuals with DMD. where at least two subgroups of individuals with DMD and as many as five subgroups of individuals with DMD were detected. The clinical factors which subdivide these subgroups of individuals with DMD is unclear.
Prediction Analysis of Individuals with DMD and Control Individuals
Prediction analysis of microarray (PAM) identified 13 genes from Tables 9 and 10) as signature genes, with an overall misclassification rate of 6%. Signature genes were identified by adjusting a threshold of fold change ≧2.2, or fold change ≦2.2, which defined the minimum number of genes that best distinguished individuals with DMD from control individuals. The PAM analysis of these 13 genes showed that, of these 34 individuals with DMD, 2 individuals were misclassified as controls, while of these 21 control individuals, 2 individuals were misclassified as individuals with DMD. Data from the cluster analysis shown in
Signaling Pathways Activated in Blood of Individuals with DMD
Based upon the genes showing ≧2 fold regulation (the 191 genes in Table 2), the signaling pathways in which these genes operated were examined. The genes in Table 2 were searched against the KEGG database of human biochemical pathways, and the pathways with the most statistically significant numbers of differentially regulated genes are shown in Table 11 (p value=significance of the number of differentially regulated genes expressed in a pathway, compared to the total number of genes currently known in that pathway; methods for calculating p value are included within the KEGG package.)
The results are shown in
The numbers of genes determined to be regulated in a particular pathway was a function of the number of genes examined. Thus, examining the 1467≧1.5 fold differentially regulated genes (Table 3) using the KEGG pathway program resulted in 3-7 times more genes in each pathway, but the pathways with the largest and most significant numbers of regulated genes for individuals with DMD, compared to control individuals, were the same as shown in
The genes regulated in blood of individuals with DMD, compared to the genes reported to be regulated in muscle of individuals with DMD, showed relatively few identical genes with the exception of utrophin. Utrophin was up-regulated in muscle in individuals with DMD (Haslett et al. cited previously), while utrophin was down-regulated in blood of individuals with DMD (Table 3). Although
Genes Regulated in Individuals with DMD were Expressed in Specific Blood Cell Types
Specific cell types from blood were not isolated in individuals with DMD or control individuals. Cell type assignment for differentially regulated genes in blood from individuals with DMD subjects was assessed using inventors' prior data Du et al., Genomics 87 (2006) 693, which is expressly incorporated by reference herein in its entirety. The genes showing ≧2 fold regulation (the 191 genes in Table 2) were mapped onto the expression profiles for different types of peripheral blood cells obtained from 3 control individuals. The genes showing ≧2 fold regulation (the 191 genes in Table 2) were expressed mainly in neutrophils, and to a lesser extent in monocytes, B cells, and CD4+ T helper cells.
Gene expression from 34 males with DMD, ranging from 3 years to 20 years with a mean age of 9±3 years, was determined (Table 8). Individuals with DMD were evaluated and had been diagnosed by a single board-certified pediatric neurologist. The diagnosis of DMD was based in part upon clinical history, examination findings, and selected muscle biopsies and was confirmed by molecular diagnostic testing.
Of the 34 individuals with DMD, 14 were administered either prednisone (PRED, n=6) or deflazacort, an oxazoline derivative of prednisolone (DEFL, n=8) at the time blood samples for gene expression evaluation were obtained. The mean duration of administration of either prednisone or deflazacort for these 14 individuals was 43.9 months. Of the 34 individuals with DMD, 20 individuals had not been administered either prednisone or deflazacort at the time blood samples for gene expression evaluation were obtained.
Sample processing and array hybridization, probe-level data analysis, and statistical analysis were identical to Example 1.
Assessment of Biological Significance of Identified Genes
Differentially regulated genes were analyzed using The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://niaid.abcc.ncifcrf.gov/) to examine co-regulation of gene pathways and biological significance. DAVID identifies over-representation of genes within particular pathways, indicating co-regulation of genes in the pathway, using the Expression Analysis Systematic Explorer (EASE) program to generate an EASE score, which is a modified Fisher Exact test score. The following DAVID default parameters were applied: minimum number of genes required per pathway or chromosome=2; maximum EASE score accepted=0.1. A DAVID Functional Annotation Clustering Report (FACR) identified similar annotations for identified probe lists. The FACR analysis addresses redundancy of different annotation databases by grouping similar annotations together, and calculates a Group Enrichment Score. The Group Enrichment Score ranks these annotation groups (in-log scale of group members' p-values in each annotation cluster) according to degree of common genes identified across annotation databases. Higher Group Enrichment Scores indicated increased level of biological significance (http://david.abcc.ncifcrf.gov/content.jsp?file=functional annotation.html).
To determine if similar transcription factors were involved in coordination of expression changes in response to corticosteroids, transcription factor binding motifs (TFBMs) that were wither over- or under-represented in differentially expressed genes were identified using the TELiS data base (http://www.telis.ucla.edu/). TELiS performs frequency analyses to compare the average number of TFBMs detected in promoters of differentially expressed genes, with the average number in non-differentially expressed genes, using a z-test.
RNA Expression Changes in Individuals with DMD not Administered Prednisone or Deflazacort, Compared to Individuals with DMD Administered Prednisone or Deflazacort
When individuals with DMD not administered prednisone or deflazacort, were compared to individuals with DMD administered either prednisone or deflazacort, expression of 524 genes was significantly different (fold change >|1.5|, unpaired t-test, p≦0.05)
Of the 524 genes, expression levels were down-regulated in 127 genes (24%), and were up-regulated in 397 genes (76%) in individuals with DMD administered prednisone or deflazacort, compared to individuals with DMD not administered prednisone or deflazacort (log-likelihood, p<0.00001) (
Promoter Sites Activated in Blood of Individuals with DMD Administered Prednisone or Deflazacort.
Using the 59 genes identified by PAM, TELiS identified 45 genes as present within its database. Using these 45 genes, TELiS identified 13 transcription factor binding motifs (TFBMs) that were significantly over-represented within the 45 regulated genes, and one TFBM that was under-represented within the 45 regulated genes. Table 12. Glucocorticoid response elements did not appear within the identified TFBMs.
Biological Significance Assessment
Using the 59 genes identified by PAM as input to DAVID, 50 genes were identified. The strongest Functional Annotation Cluster (enrichment score=5.9) identified by DAVID reflected antibiotic and antimicrobial response functions (p-value range 4.30−9 to 2.20−3). Table 13. Within this list manual curation identified genes determined to be up-regulated in individuals with DMD administered prednisone or deflazacort, involved in iron trafficking (lipocalin, lactotransferrin, haptoglobin, cathepsin G, azurocidin, elastin), and involved in chondroitin sulfate synthesis (chondroitin β1,4 N-acetylgalactosaminyltransferase).
RNA Expression Changes in Blood in Individuals with DMD Administered Deflazacort Compared to Individuals with DMD Administered Prednisone
Expression of 508 genes was significantly different in individuals with DMD administered deflazacort compared to individuals with DMD administered prednisone (fold change ≧|1.5|, unpaired t-test, p≦0.05) (
The expression profile of the 2 individuals that were initially administered prednisone and later administered deflazacort was examined to determine whether the expression profiles for these individuals would resemble either of the previous groups. Using the 508 gene list, these 2 individuals had expression patterns similar to individuals administered prednisone, using either unsupervised hierarchical clustering (
Functional Annotation Clustering
Using the 496 gene set, the strongest Functional Annotation Cluster (enrichment score=7.49) identified by DAVID reflected intracellular signaling and signal transduction (p-value range 1.70−10 to 1.40−6) (Table 14). Further manual curation identified genes in the 496 gene set involved in adipose formation, retinoic acid signaling, and NFKB pathways.
Overlap Between Comparisons
Eighteen genes were identified as differently regulated in both individuals administered prednisone or deflazacort, compared to individuals not administered prednisone or deflazacort (18/524, 0.03%), and in individuals administered deflazacort compared to individuals administered prednisone (18/508, 0.04%) (Table 15). Nucleic acids 1702, 1848, 1750, 1576, 1738, 1711, 1650, 1686, 1675, 1675, 1807, 1664, 1590, 1736, 1596, 1688, 1667, and 1660.
Homo sapiens cDNA:
Homo sapiens full length insert
Homo sapiens cDNA FLJ37655
Homo sapiens brain my041
Homo sapiens B lymphocyte
Homo sapiens EEN-B2-L4 mRNA,
Homo sapiens cDNA: FLJ23573 fis,
Homo sapiens full length insert cDNA
sapiens cDNA FLJ37655 fis, clone
sapiens partial IGVH4 gene for
Homo sapiens brain my041 protein
Homo sapiens B lymphocyte
sapiens cDNA clone IMAGE: 2686541
sapiens cDNA FLJ38183 fis, clone
sapiens PRO2221 mRNA, complete
Homo sapiens brain my038 protein
sapiens cDNA clone IMAGE: 2456964
Homo sapiens mRNA; cDNA
Homo sapiens mRNA; cDNA
sapiens cDNA clone IMAGE: 3278832
sapiens cDNA clone IMAGE: 3825757
Homo sapiens cDNA clone UI-E-EO1-
Homo sapiens mRNA; cDNA
S. cerevisiae)
Homo sapiens cDNA:
Homo sapiens full length
Homo sapiens cDNA
Homo sapiens brain my041
Homo sapiens B lymphocyte
sapiens cDNA clone
sapiens translocase of inner
sapiens astrotactin 2, mRNA
Homo sapiens cDNA
Homo sapiens PRO2221
Homo sapiens brain my038
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
sapiens cDNA clone
sapiens cDNA clone
Homo sapiens hypothetical
sapiens lymphocyte-
Homo sapiens cDNA
Homo sapiens , Similar to
sapiens cDNA clone
Homo sapiens mRNA; cDNA
sapiens cDNA clone
sapiens]
Homo sapiens cDNA
Homo sapiens cDNA
Homo sapiens pregnancy-
Homo sapiens clone
Homo sapiens cDNA
Homo sapiens , clone
sapiens cDNA clone
Homo sapiens histone 1,
Homo sapiens cDNA clone
Homo sapiens cDNA
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens cDNA:
sapiens cDNA clone
sapiens]
sapiens cDNA clone
sapiens cDNA, mRNA
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA
Homo sapiens cDNA:
Homo sapiens cDNA clone
sapiens cDNA, mRNA
Homo sapiens mRNA; cDNA
sapiens cDNA clone
sapiens cDNA clone
Homo sapiens cDNA clone
sapiens cDNA clone
sapiens cDNA clone
sapiens cDNA clone
sapiens cDNA clone
Homo sapiens partial IGVH3
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens toll-like
Homo sapiens mRNA; cDNA
sapiens cDNA clone
sapiens cDNA clone
Homo sapiens cDNA
Homo sapiens PRO1847
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
sapiens]
sapiens cDNA 3′, mRNA
H. sapiens c6.1A mRNA.
Homo sapiens cDNA:
sapiens cDNA clone
Homo sapiens genomic DNA,
sapiens cDNA 3′, mRNA
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens mRNA for
Homo sapiens cDNA clone
Homo sapiens cDNA 3′ end,
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens KIAA0971
sapiens cDNA clone
sapiens ring finger protein 44
Homo sapiens HSNFRK
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens , SKAP55
sapiens PAC clone RP5-
Homo sapiens cDNA clone
Homo sapiens mRNA; cDNA
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA:
Homo sapiens cDNA
Homo sapiens cDNA clone
Homo sapiens PRO1847
Homo sapiens IDN4-GGTR7
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens IgH VH gene
Homo sapiens cDNA clone
sapiens cDNA clone UI-H-
sapiens cDNA clone UI-H-
Homo sapiens cDNA clone
Drosophila) (LOC343574),
Homo sapiens PRO1412
sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens sorting nexin 2
Homo sapiens mRNA; cDNA
Homo sapiens mRNA; cDNA
sapiens cDNA clone
Homo sapiens cDNA clone
sapiens cDNA clone
Homo sapiens mRNA; cDNA
Homo sapiens, clone IMAGE: 4695648, mRNA
Homo sapiens, clone IMAGE: 5745627, mRNA
Homo sapiens, clone IMAGE: 4272847, mRNA
Homo sapiens (human)
Homo sapiens, clone IMAGE: 4695648, mRNA
Homo sapiens, clone IMAGE: 5745627, mRNA
Homo sapiens, clone IMAGE: 4272847, mRNA
Homo sapiens (human)
Other variations or embodiments of the invention will also be apparent to one of ordinary skill in the art from the above figures and descriptions. Thus, the forgoing embodiments are not to be construed as limiting the claim scope.
Applicants incorporate by reference the material contained in the accompanying computer readable Sequence Listing identified as Sequence_Listing.txt, having a file creation date of Oct. 13, 2009 3:54:51 P.M. and file size of 115 KB.
This application claims priority from U.S. patent application Ser. No. 61/105,577 filed Oct. 15, 2008 with a petition for revival in progress, and co-pending U.S. patent application Ser. No. 61/105,565 filed Oct. 15, 2008, each incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61105577 | Oct 2008 | US | |
| 61105565 | Oct 2008 | US |
