Patent Application
20090010908
The invention relates to chronic fatigue syndrome/myalgic encephalitis, and in particular to materials and methods for its diagnosis and treatment.
Chronic fatigue syndrome (CFS), also known as Myalgic Encephalomyelitis (ME), is an acquired disorder with long term disability1. The illness affects both children and adults and is characterised by persistent or relapsing fatigue of sufficient severity to interfere with normal function. In addition, patients experience impairment in short term memory and concentration, muscle pain and prolonged post-exertional malaise2. The estimated overall prevalence of CFS/ME in the community is approximately 0.2-0.4% in adults and 0.07% in adolescents and children3.
The pathogenesis of CFS/ME is unknown. Diagnosis of CFS/ME according to the internationally accepted definition (modified CDC criteria) is essentially based on exclusion of known medical and psychiatric diseases2. As yet, there is no specific or sensitive diagnostic test that positively establishes and/or supports the clinical diagnosis of CFS/ME.
Nevertheless, in 2002, following the publication of a UK Government Working Party report, Professor Sir Liam Donaldson confirmed that CFS/ME is a debilitating and distressing condition affecting many people which should be classed alongside other diseases such as multiple sclerosis and motor neurone disease”.
A significant proportion of patients report antecedent history of community acquired viral or bacterial infections4,5,6. In addition, it is clear that patients with CFS/ME have a hypoactive hypothalamic-pituitary-adrenal (HPA) axis with altered neuroendocrine regulation affecting neurotransmitters such as monoamines (norepinephrine, serotonin and dopamine) and acetylcholine in the central nervous system4. Recent studies have demonstrated that viruses can affect neurotransmitter functions7,8.
At the cellular level, fatigue has been linked with alterations in the cell membrane ion-channel traffic and ATPase system9. ATPases are also linked with neurotransmitter release (e.g. dopamine)10 and cellular energy metabolism via creatine phosphatase. Increased ATPase activity has been reported in muscle biopsies from patients with CFS11,12. Previous work11,13 has raised the possibility that patients with CFS may have an ion channel dysfunction. This dysfunction might be induced by changes in the ion channel function, neurotransmitters involved in “gating” the channel or by a shift in the balance of the cellular “energy charge”, i.e. the ratio between ATP and ADP that is normally a function of the ATPase activity.
One previous study43 has identified a number of genes differentially expressed between PBMCs from CFS/ME patients and healthy controls. A number of the genes identified are implicated in various aspects of immunological function, from which the authors concluded that some kind of immunological dysfunction may be involved in pathogenesis of CFS/ME. The authors were unable to relate the observed expression patterns to any functional model of disease etiology or pathology, and did not suggest that any of the genes which they identified could serve as useful biomarkers for CFS/ME. Their conclusions are consistent with previous studies which have suggested immunological abnormality in CFS/ME (Refs. 44 to 48) but the precise mechanism of immunological dysfunction in CFS/ME has not been established.
The present inventors have identified a number of genes which are expressed at abnormal levels in patients affected by CFS/ME as compared to normal healthy individuals. In contrast to the earlier studies described above, the present inventors have been able to use the expression patterns of these genes to establish functional models of various aspects of the pathology of CFS/ME, which explain many of the symptoms observed in affected individuals. These provide a rational basis for classifying CFS/ME patients according to the biochemical lesion underlying their symptoms and enable appropriate targeted therapies to be provided for the first time.
The genes identified in the present invention provide objective disease markers that may be used in diagnostic tests to support the diagnosis of CFS/ME or in other applications. For example, the tests may enable deselection of inappropriately diagnosed patients who have an alternative diagnosis for their fatigue symptoms. The tests may also be used to classify patients according to the particular biochemical basis for their symptoms. In turn, this may enable a clinician to identify therapies which would be appropriate and rule out those which would not. The tests may also be applied as an outcome measure for interventional trials in CFS/ME, not least because no specific treatment is currently considered to be effective in the patient population defined by the clinical criteria. Further, the tests may offer support to CFS/ME patients for their claims for disability and insurance. Finally, depending on the specificity and sensitivity of the test, one may apply it to screen patients with symptoms of chronic fatigue to identify or exclude CFS/ME. Given the fact that chronic fatigue as a symptom is ten times commoner in the population (2%) than CFS/ME, an estimated size of the market for a screening test is at least 10 million in the UK alone.
Thus the present invention provides a method for investigating whether a test subject is affected by chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), the method comprising providing a biological sample from the subject and determining the level in the sample of a biomarker for CFS/ME, wherein the biomarker is an expression product of a gene shown in Table 1. Genes in Table 1 have been found to be overexpressed in CFS/ME compared to unaffected individuals.
The method may comprise determining the level, in the sample, of a plurality of biomarkers (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more biomarkers), wherein each of the biomarkers is an expression product of a gene shown in Table 1.
The method may comprise the step of determining the level in the sample of one or more further biomarkers, being expression products of one or more genes from Table 6. Indeed, an expression product from any one of the genes of Table 6 may be used as a biomarker for CFS/ME, either alone or in combination with other genes of Table 6 or of Tables 1 to 5.
The method may be considered to provide an expression profile of the one or more biomarkers for CFS/ME, for the test subject, at the time of sampling. By “expression profile” is meant a set of data relating to the level of expression of one or more of the relevant biomarkers in a test subject, in a form which allows comparison with comparable expression profiles (e.g. from affected and/or unaffected individuals), in order to assist in the determination of whether or not the subject is affected by CFS/ME.
The method typically involves correlating the results obtained with a probability that the subject is affected by CFS/ME. In order to assist with this correlation, the method of the invention may comprise the step of comparing the expression profile for the test subject with one or more “reference” expression profiles, that is to say one or more expression profiles characteristic of unaffected subjects (i.e. subjects not suffering from CFS/ME), and/or one or more expression profiles characteristic of affected subjects (i.e. subjects not suffering from CFS/ME). Thus the level of expression of one or more of the said biomarkers in the test subject is typically compared with that characteristic of affected and/or unaffected individuals.
The reference expression profiles may be profiles previously derived from healthy or affected individuals, or may be artificial profiles which display expression levels of the relevant biomarkers which are characteristic of the relevant group. For example, they may be computer-generated from a plurality of profiles previously derived from appropriate individuals. The profile(s) characteristic of affected subjects may be divided into subgroups, e.g. according to their symptoms, as described in more detail below.
Taken alone, the expression profile for the test subject may not provide an absolute diagnosis of CFS/ME. Normally, a clinician will also take account of the physical and/or psychological symptoms of the subject in order to reach a diagnosis. However the expression profile generated by the methods of the invention provides useful data to help the clinician confirm or reject a preliminary diagnosis based on physical and psychological symptoms alone. A finding that one or more of these genes is upregulated in a particular individual may therefore provide support for a diagnosis of CFS/ME.
By “upregulated” or “overexpressed” is meant that the gene in question shows at least a two fold increase in expression at the level of mRNA and/or protein as compared to the level observed in unaffected (preferably healthy) individuals. If desired, the level of overexpression required to regard a result as positive may be set higher than this, e.g. a three, four, five, six, seven, eight, nine or ten fold increase as compared to unaffected individuals, or higher if required.
The genes in Table 1 not only provide biomarkers for CFS/ME, they also provide the first evidence of specific biochemical pathways which may be dysregulated in CFS/ME. They can be clustered into a number of subgroups, based on their involvement in the same or related biochemical pathways whose dysregulation is likely to play a role in either the underlying cause or the symptoms of the disease state. Previously it has not been possible to identify such pathways, which is a primary reason why no generally-recognised therapies exist for CFS/ME. The subgroups of genes identified by the present inventors provide an explanation for many of the symptoms displayed by those affected by CFS/ME, suggest rational therapies for the condition, and provide means for monitoring the efficacy of any therapy administered.
Without wishing to be bound by any particular theory, the inventors believe that CFS/ME is not a genetic disease caused by a single or multiple gene defects. Rather, they believe CFS/ME to be an acquired condition where there is a shift in the functional systems of a select number of genes regulating specific biological functions (e.g. infection and immunity, cell membrane function and cell cycle). Based on the expression patterns they have observed, they have established a model of “hub” and “network” genes which emphasises the interrelation of these key genes, with the hub genes being the control centre and the network genes being largely the effector arm of this functional system. This set of hub and network genes defines the functional shift in the biological systems of patients who continue to have symptoms due to CFS/ME.
Thus the genes of Table 1 are subdivided into “hub” genes and “network” genes. Essentially, in any particular pathway the hub gene(s) can be considered to lie upstream of the network genes, in that dysregulation of the hub gene is likely to lead (directly or indirectly) to dysregulation of the downstream genes. In Table 1, the hub genes are shown in section A, and the network genes in section B.
Certain genes which appear to be highly significant (e.g. based on the difference in their expression between the disease state and the normal state) have been allocated to the “hub” group even where no related set of downstream “network” genes is specifically identified.
Exemplary groups of genes identified in Table 1 are detailed below.
Defensin α1, CXCR4 and lactotransferrin (LTF) are all involved in the response to intracellular infection. The network genes associated with these hub genes include defensin α4, integrin α2B, integrin β3, arginase 1, arginase 2, thrombospondin 1, membrane associated protein 17 (MAP 17), Charcot Leyden Crystal Protein (CLC) and chondroitin sulphate proteoglycan 2 (versican). These genes are shown in Table 2.
Haemoglobin γ (foetal haemoglobin) is part of the oxidative stress response pathway, and is only expressed under conditions of oxidative stress. Network genes associated with the oxidative stress pathway include other haemoglobin genes including haemoglobin alpha 1, prostaglandin-endoperoxide synthase 1 and prostaglandin-endoperoxide synthase 2. These genes are shown in Table 3. However it will be noted that Table 6 includes genes for haemoglobins alpha, beta, gamma and delta. Any one of the haemoglobin genes of Table 6 may be used as a biomarker for CFS/ME, and may be used as well as, or in place of haemoglobin gamma or alpha if desired. For simplicity, though, reference will be made primarily to haemoglobin gamma.
Serine/threonine kinase 17B (STK17B) is implicated in apoptosis, as are the network genes caspase 1, dynamin 1-like, and phosphatidyl serine binding protein. These genes are shown in Table 4.
The MHC class II gene HLA-DRβ4 is upregulated in CFS/ME implying a shift from antigen presentation by MHC class I to MHC II presentation. Associated network genes include HLA-DQβ1 and the immunoglobulin heavy chain γ3 (IgG3). These genes are shown in Table 5. IgG1 may be used as a biomarker as well as, or in place of, IgG3.
Methods for investigating whether a test subject is affected by CFS/ME may therefore comprise determining the level of at least one gene from each of at least two of Tables 2, 3, 4 and 5, and preferably from each of three or four of said Tables. In preferred embodiments, at least one gene from each said Table is a hub gene.
Preferred genes of Table 1 are defensin α1, haemoglobin γ, CXCR4, tubulin beta 1 and HLA DRβ4. The method may involve testing expression of any one, two, three, four or all five of these genes, and optionally further hub genes of Table 1A.
Additionally or alternatively, the method may involve testing expression of one or more of TSP 1, caspase 1 and/or IgG3.
The method may also comprise the step of determining the level of an expression product of prostaglandin D2 synthase in sample. In contrast to the genes of Table 1, prostaglandin D2 synthase is found to be downregulated in individuals suffering from CFS/ME as compared to normal controls. Thus a finding that prostaglandin D2 synthase is downregulated in a particular individual may provide support for a diagnosis of CFS/ME.
By “downregulated” or “underexpressed” is meant that prostaglandin D2 synthase shows at least two fold higher expression at the level of mRNA and/or protein in unaffected (preferably healthy) individuals than is observed in the test subject. If desired, the level of downregulation required to regard a result as positive may require the level found in unaffected individuals to be three, four, five, six, seven, eight, nine or ten fold higher than in affected individuals.
The method may further involve the step of determining the level, in a biological sample from the subject, of the peptide QYNAD, as described in more detail below. The level of the peptide in the sample may form part of the expression profile established for the subject. An elevated level of the peptide as compared to unaffected individuals may be indicative of CFS/ME. By an “elevated” level is meant at least twice the level found in unaffected healthy controls, and preferably at least 5 times or at least 10 time the level found in unaffected healthy controls.
It will be appreciated that individual patients who each satisfy the criteria for CFS/ME may nevertheless present a range of very different symptoms. Some of these can be explained by the groups of genes identified herein.
For example, CFS/ME has previously been suggested to involve immune dysfunction. Some CFS/ME sufferers are particularly prone to infection by viruses (e.g. influenza) and other pathogens; indeed some suffer recurrent infections. Others are affected by atopic/allergic symptoms. These symptoms may be associated with the apparent shift from Type I to Type II antigen presentation by the MHC, which could impair the body's ability to deal efficiently with infections and could also exacerbate allergy/atopy.
Some CFS/ME sufferers describe their body as feeling prematurely aged, and can display restricted mobility characteristic of much older individuals. This might be explained by excessive apoptosis, particularly in the central nervous system. Therefore patients having these symptoms might be expected to show increased expression of serine/threonine kinase 17B (STK17B) and its associated network genes.
Oxidative stress is also a cause of apoptosis, particularly in the nervous system. Thus patients with increased expression of haemoglobin γ and its associated network genes may also show, or be at risk of, accelerated programmed cell death (increased level of apoptosis), particularly neuronal apoptosis. Features of oxidative stress and neuronal apoptosis may include ageing, cognitive impairment and chronic pain.
The genes of Table 2 are implicated in cellular protection (“defence”) against viral or bacterial infections. Thus an overexpressed defensin gene in CFS/ME would be consistent with immune activation and correlate with symptoms of recurrent influenza-type symptoms, sore throat and lymph node enlargement experienced by patients with CFS/ME.
In a further aspect, the invention therefore provides a method for classifying a subject affected by CFS/ME, the method comprising providing a biological sample from the subject and determining the level in the sample of a biomarker for CFS/ME, wherein the biomarker is an expression product of a gene shown in one or more of Tables 2 to 5.
Preferably the biomarker is a hub gene from one of Tables 2, 3, 4 or 5. More preferably, the method comprises determining the expression level of hub genes from each of two, three or all four of Tables 2, 3, 4 or 5. The method may also comprise determining network genes from one, two, three or all four of Tables 2, 3, 4 and 5.
The individual can then be assigned to a subgroup of CFS/ME, dependent on which group or groups of genes are found to be upregulated. Thus, for example, the subject may be classified as being affected by one or more of oxidative stress, excessive apoptosis, and immunological dysregulation (MHC I to II shift). The assignment step may involve comparing the expression profile obtained from the subject with one or more expression profiles characteristic of individuals previously assigned to one or more subgroups of CFS/ME.
It will be appreciated that the classification may be performed using the same expression profile as that established for determining whether the subject is affected by CFS/ME.
Individuals in which these particular pathways are dysregulated may be suitable for treatment by the methods described below. Therefore in a further aspect the invention provides a method of determining whether an individual affected by CFS/ME is suitable for treatment using such a therapy. The method comprises determining whether the individual is affected by one or more of oxidative stress, excessive apoptosis, and immunological dysregulation as described above, and optionally prescribing a suitable treatment depending on the outcome. These treatments are described in detail below.
Expression of individual biomarkers for CFS/ME (genes from Tables 1 to 5) may differ slightly between independent samples, leading to slightly different expression profiles for individual samples. However, these particular genes may provide a characteristic pattern of expression (expression profile) in an affected individual (i.e. one suffering from CFS/ME) that is recognisably different from that in an unaffected individual (i.e. one not suffering from CFS/ME).
By creating a number of expression profiles from a number of known affected and unaffected samples, it is possible to create a library of profiles for both sample types. The greater the number of expression profiles, the easier it is to create a reliable characteristic expression profile standard (i.e. including statistical variation) that can be used as a control in a diagnostic assay. Thus, a standard profile may be one that is devised from a plurality of individual expression profiles and devised within statistical variation to represent either the affected or unaffected profile.
The determination of the expression profile may be computerised and may be carried out within certain previously set parameters, to avoid false positives and false negatives.
The computer may then be able to provide an expression profile standard characteristic of an affected sample and a normal sample. The determined expression profiles may then be used to classify test samples as affected or unaffected as a way of diagnosis.
Thus, in a further aspect the invention provides a method of creating a library of expression profiles for use in determining whether an individual is affected by CFS/ME, the method comprising
(a) providing biological samples from a plurality of individuals affected by CFS/ME, and determining the level in each sample of one or more biomarkers for CFS/ME to create a plurality of expression profiles from affected individuals;
(b) providing biological samples from a plurality of individuals not affected by CFS/ME, and determining the level in the sample of said one or more biomarkers for CFS/ME to create a plurality of expression profiles from unaffected individuals;
wherein the biomarkers comprise expression products of one or more genes shown in Table 1.
Typically, the method comprises the step of retrievably storing each of the expression profiles on a computer data carrier, in order to create a database of expression profiles for both affected and unaffected individuals.
The invention further provides an expression profile database, comprising a plurality of expression profiles of biomarkers for CFS/ME from affected and unaffected individuals, wherein the biomarkers comprise expression products of one or more genes shown in Table 1.
It will be appreciated that the profiles may be classified according to their expression levels of the various groups of genes shown in Tables 2 to 5 as already described. The combinations of biomarkers analysed in establishing the expression profiles may therefore be chosen as described above.
The expression profiles may comprise data relating to the level, in biological samples from the subjects, of the peptide QYNAD. If required, the methods may therefore comprise the step of determining the level of this peptide in biological samples from the subjects.
At present there are no generally acknowledged treatments for CFS/ME. One problem which has faced researchers attempting to develop and evaluate suitable therapies is the lack of objective testable criteria to determine their efficacy. The present invention provides a useful tool for monitoring the efficacy of an interventional treatment for CFS/ME, by studying the effect of the therapy, over time, on an individual's expression profile of the biomarkers described in Tables 1 to 5. The methods described may therefore be used to evaluate the effectiveness of a test treatment, e.g. by testing it on a population of subjects affected by CFS/ME, or to investigate an individual's response to a particular therapy, to see whether or not they are responding appropriately.
Thus in a further aspect the invention provides a method of determining the efficacy of a treatment for CFS/ME comprising the steps of:
(a) providing a biological sample from a subject affected by CFS/ME who has been subjected to said treatment,
(b) determining the level in said sample of one or more biomarkers for CFS/ME to create an expression profile for said subject, and
(c) comparing said expression profile with
i) a comparable expression profile obtained from said test subject before initiation of said treatment, and/or
ii) a comparable expression profile obtained from said test subject at an earlier stage of said treatment, and/or
iii) a comparable expression profile characteristic of a subject who is unaffected by CFS/ME,
wherein the one or more biomarkers for CFS/ME are expression products of one or more genes shown in Tables 1 to 5. Preferred combinations of biomarkers have already been described in relation to earlier aspects of the invention.
In general, a treatment may be considered to be effective if the subject's expression profile after treatment shows that the expression level of one or more of the biomarkers is reduced compared to its level before treatment, or its level earlier in a course of the treatment. Preferably the subject's expression profile after treatment approaches a profile characteristic of an individual unaffected by CFS/ME. That is to say, the expression level of each of the biomarkers falls within the normal range found in unaffected individuals.
Some treatments may be targeted to particular pathways known to be dysregulated in CFS/ME. In such cases, it may be desirable to follow the effect of the treatment on the expression levels of genes in the pathway likely to be modulated by that treatment. For example, a treatment which is intended to reduce oxidative stress, apoptosis or immune dysfunction (characterised by MHC shift) may have a particular effect on the expression levels of the genes in Tables 3, 4 and 5 respectively. The biomarkers chosen to study the effects of the treatment may be chosen accordingly. Preferred combinations of biomarkers from these Tables have already been described.
The method may further comprise determining the effect of the treatment on the level of the peptide QYNAD in a biological sample from the test subject.
In the diagnostic and analytical methods described herein, the expression levels of the chosen biomarker(s) for CFS/ME are preferably determined using peripheral blood mononuclear cells (PBMCs) from the test subject. Therefore the biological sample used to establish the expression profile is preferably a blood sample or cells isolated from a blood sample. If desired, the method may comprise the steps of enriching PBMCs in the sample, or isolating PBMCs from the sample.
It will be understood that the methods described are generally performed on a biological sample which has been isolated from the test subject. The method may, but need not, comprise the actual step of isolating the sample from the test subject, e.g. by taking a blood sample.
Typically, the expression level of each of the biomarkers is determined by contacting the sample with a binding agent capable of binding specifically to an expression product of the genes encoding that biomarker (a gene of Table 1, 2, 3, 4, 5 or 6). Binding between the agent and an expression product present in the sample is then determined. If it is desired to determine the expression level of more than one gene, then the sample may be contacted with a plurality of binding agents, either simultaneously or sequentially, each binding agent being capable of binding specifically and individually to an expression product of one of the biomarker genes.
Binding agents capable of binding to nucleic acid expression products (e.g. mRNA, pre-mRNA) are typically nucleic acid primers or probes having a sequence of, or complementary to, a portion of the nucleic acid expression product. This enables the binding agent to hybridise under suitable conditions with the nucleic acid expression product itself, e.g. in a Northern blot or in situ hybridisation assay, or to a cDNA copy of the nucleic acid expression product, e.g. in a RT-PCR assay, Southern blot or microarray assay.
Binding agents capable of binding to polypeptide expression products include ligands and receptors for the polypeptide in question. Particularly preferred examples of binding agents are antibodies specific for (e.g. raised against) the desired polypeptide, or fragments thereof comprising an antigen binding site. These may be used in a variety of immunological assay methods, including Western blots and ELISA assays, as well as in microarray assays.
It may be desirable to isolate expression products or particular fractions thereof (e.g. total RNA, mRNA, total protein, soluble proteins or membrane proteins) from the sample before contacting them with the binding agent. The extent to which this is necessary will vary depending on the chosen assay method.
Simple immunological assays (such as ELISA assays) or PCR-based assays using ten biomarkers or less (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers) are particularly preferred, because they can readily be adapted to analyse large numbers of samples in a relatively short space of time, at relatively low cost. Such assays are also well-suited to automation. Preferred markers and combinations of markers are as described elsewhere in this specification.
The diagnostic and analytical methods described above may also comprise determining the level, in a biological sample from the subject, of the peptide QYNAD. It is believed that the peptide may be found in serum and cerebrospinal fluid (CSF) in most or all of the population, but it is found at elevated levels in subjects suffering from inflammatory and immunological disorders of the nervous system (such as multiple sclerosis and Guillain-Barre syndrome). The present inventors have now established that it is also found at significantly elevated levels in subjects suffering from CFS/ME.
The biological sample used for determining the peptide level may be a blood sample (or serum derived therefrom). Thus, conveniently, the same blood sample may be used for determining levels of the peptide and the other biomarkers of CFS/ME. Alternatively, a different sample may be used, in particular, a sample of cerebrospinal fluid (CSF) where available.
The level of the peptide may be determined as described by Brinkmeier et al.16, e.g. by gel filtration chromatography. Alternatively conventional immunoassays such as ELISAs or Western blots may be used, employing antibodies raised against the peptide.
The assay for the peptide is generally performed on a sample isolated from the test subject. The method of the invention need not comprise the step of actually isolating the sample from the test subject.
In a further aspect, the invention provides a kit for use in a diagnostic or analytical method as described herein, the kit comprising a plurality of binding agents, each capable of binding specifically and individually to an expression product of one of the genes of Table 1, or the peptide QYNAD. Thus the kit includes binding agents specific for expression products of two or more genes of Tables 1, or at least one of the genes of Table 1 and the peptide QYNAD. The binding agents provided in the kit may be capable of binding specifically and individually to expression products of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more of the genes of Table 1 and optionally the peptide QYNAD.
The kit is suitable for use in the methods of the invention described in this specification, and may comprise instructions for performing one or more methods of the invention.
The binding agents may be immobilised on one or more solid supports. Discrete supports may each carry only one type of binding agent. For example, distinct populations of beads may each carry one type of binding agent. Alternatively, a single support may carry more than one type of binding agent. Indeed, one support (e.g. a microarray chip) may carry all of the different types of binding agent provided with the kit.
In addition, the kit may comprise one or more binding agents capable of binding specifically to an expression product of a control gene which is not differentially expressed between individuals affected and unaffected by CFS/ME. The level of expression from this control gene may be measured in order to assist in quantification of the expression products of the genes of Table 1, and/or for quality assurance of an assay performed using the kit. Preferably a control gene is chosen which is constitutively expressed in the cells of the biological sample (i.e. always expressed, at substantially the same level, under substantially all conditions). Such genes are often referred to as “housekeeping” genes. Examples include glyceraldehyde phosphate dehydrogenase (GAPDH), β-actin, and abl (ableson tyrosine kinase).
The kit may comprise yet further binding agents capable of binding to expression products of other biomarker genes or control genes. However, in preferred embodiments, the kit comprises binding agents for expression products of less than 1000 different genes, e.g. less than 500 different genes, less than 100, less than 50, less than 40, less than 30, less than 20, or less than 10 different genes.
As explained above, the groups of genes identified by the present inventors also suggest specific therapies for CFS/ME.
Patients displaying signs of oxidative stress may be treated with an anti-oxidant. Thus the invention provides a method of treating CFS/ME in an individual suffering therefrom, comprising administering an effective amount of an anti-oxidant.
Therapeutically effective amounts of a corticosteroid and/or minocyline may also be administered to the subject in conjunction with the anti-oxidant. As will be appreciated, the individual active agents may be administered individually (in two or more separate compositions) or together (in the same composition).
The invention further provides the use of an anti-oxidant in the preparation of a medicament for the treatment of CFS/ME. The medicament may be formulated for administration in conjunction with a corticosteroid and/or minocycline. Alternatively, the medicament may comprise a corticosteroid and/or minocycline.
Examples of suitable anti-oxidants include coenzyme Q10 and inhibitors of cyclooxygenase (COX) enzymes, particularly COX II enzymes, such as celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide).
Patients showing evidence of abnormal apoptosis may be treated with minocyline, which is an inhibitor of caspase 1, shown here to be upregulated in individuals affected by CFS/ME. Minocyline has previously been suggested for treatment of various neurological disorders including stroke, multiple sclerosis, spinal cord injury, Parkinson's disease, Huntington's disease and amylotrophic lateral sclerosis—see Wee Young et al., Lancet Neurology, 2004, 744-751 for review.
Thus the invention provides a method of treating CFS/ME in an individual suffering therefrom, comprising administering an effective amount of minocycline.
Therapeutically effective amounts of a corticosteroid and/or an anti-oxidant may also be administered to the subject in conjunction with the minocycline. As will be appreciated, the individual active agents may be administered individually (in two or more separate compositions) or together (in the same composition).
The invention further provides the use of minocycline in the preparation of a medicament for the treatment of CFS/ME. The medicament may be formulated for administration in conjunction with a corticosteroid and/or an anti-oxidant. Alternatively, the medicament may comprise a corticosteroid and/or an anti-oxidant.
Oxidative stress often gives rise to apoptosis. Therefore it may be advisable to treat patients showing signs of oxidative stress with an apoptosis treatment such as minocycline as a precautionary measure if they do not already show upregulation of genes involved in apoptosis.
Patients showing signs of immune dysfunction, and particularly the Type I to II shift of MHC expression, may benefit from treatment with a corticosteroid. This would address the hypocortisolism previously reported in some CFS/ME patients.
Thus the invention provides a method of treating CFS/ME in an individual suffering therefrom, comprising administering an effective amount of a corticosteroid.
Therapeutically effective amounts of minocycline and/or an anti-oxidant may also be administered to the subject in conjunction with the corticosteroid. As will be appreciated, the individual active agents may be administered individually (in two or more separate compositions) or together (in the same composition).
The invention further provides the use of a corticosteroid in the preparation of a medicament for the treatment of CFS/ME. The medicament may be formulated for administration in conjunction with minocycline and/or an anti-oxidant. Alternatively, the medicament may comprise minocycline and/or an anti-oxidant.
An example of a suitable corticosteroid is hydrocortisone. Others include dexamethasone and prednisone.
The invention further provides pharmaceutical compositions suitable for the treatment of CFS/ME. Thus there is provided a pharmaceutical composition comprising a therapeutically effective amount of minocycline in combination with a therapeutically effective amount of a corticosteroid and/or an anti-oxidant and a pharmaceutically acceptable carrier.
Also provided is a pharmaceutical composition comprising a therapeutically effective amount of a corticosteroid in combination with a therapeutically effective amount of minocycline and/or an anti-oxidant, and a pharmaceutically acceptable carrier.
Also provided is a pharmaceutical composition comprising a therapeutically effective amount of an anti-oxidant in combination with a therapeutically effective amount of a corticosteroid and/or minocycline, and a pharmaceutically acceptable carrier.
As described above, a particular treatment may be determined for any individual based on their expression profile of CFS/ME biomarkers. For example, an individual showing upregulation of oxidative stress pathway genes may be particularly suitable for treatment with an anti-oxidant. However it may not be convenient or necessary to establish a suitable expression profile before beginning treatment.
Therefore it may be desirable to administer one, two or all three of the treatment types described above to an individual affected by CFS/ME without recourse to expression profiling.
CFS is typically diagnosed using the modified CDC criteria described by Fukuda et al.2. All other conditions or diseases which could explain a patient's symptoms are first excluded. Having done this, CFS/ME is diagnosed if the patient has been affected by 6 months or longer of persistent relapsing or persistent fatigue accompanied by four or more concurrent symptoms including impaired memory severe enough to affect normal daily function, sore throat, tender lymph nodes, muscular or joint pain, new headaches, unrefreshing sleep and post-exertional malaise lasting for more than 24 hours. For the purposes of this specification, individuals satisfying these criteria are considered to be affected by CFS/ME.
The genes identified in Table 1 provide biomarkers which may be used in diagnostic assays to support, confirm, or refute a diagnosis of CFS/ME. With the knowledge of this set of genes, it is possible to devise many methods for determining a suitable expression profile of one or more CFS/ME biomarkers in a particular test sample.
Typically, the method involves contacting expression products from the sample with a binding agent capable of binding to an expression product of a gene identified in Table 1. The expression product may be a transcribed nucleic acid sequence or an expressed polypeptide.
The transcribed nucleic acid sequence may be mRNA or pre-mRNA. Alternatively, the expression product may also be cDNA produced from said mRNA. The binding member may a nucleic acid having a sequence complementary to that of the RNA or cDNA which is consequently capable of specifically binding to the transcribed nucleic acid or cDNA under suitable hybridisation conditions, e.g. by Northern blotting, in situ hybridisation, or Southern blotting.
Such protocols may use probes of at least about 20-80 bases in length. The probes may be of 100, 200, 300, 400 or 500 bases in length or more. Binding assays may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989 or later editions).
RT-PCR procedures (including quantitative PCR procedures) may also be used to analyse the presence or amount of mRNA or precursor mRNA in a given sample. A suitable primer having at least 15 to 20 bases complementary to the desired mRNA or precursor mRNA sequence will typically be used to prime cDNA synthesis. Alternatively a poly-T primer (optionally comprising one or more random nucleotides at the 3′ end) may be used to prime cDNA synthesis from all mRNA in the sample.
Subsequently, a segment of the cDNA is amplified in a PCR reaction using a pair of nucleic acid primers, each typically having at least 15 to 20 bases complementary to the desired RNA sequence. The skilled person will be able to design suitable probes or primers based on the publicly available sequence data for the genes in question (see Table 1 for suitable accession numbers).
Where the expression product is the expressed polypeptide, the binding member is preferably an antibody raised against or otherwise specific for the desired polypeptide, or any other molecule comprising the antigen binding site from such an antibody.
The skilled person will realise that other binding agents may be used as appropriate. Suitable agents may include naturally-occurring ligands and receptors for the desired polypeptide, aptamers, etc. For example, aptamers are nucleic acid molecules (typically DNA or RNA), selected from libraries on the basis of their ability to bind other molecules. Aptamers have been identified which can bind to other nucleic acids (by means other than conventional Watson-Crick base pairing), proteins, small organic compounds, and even entire organisms.
The binding agent (e.g. a nucleic acid probe or antibody) may be fixed to a solid support. The expression products may then be passed over the solid support, thereby bringing them into contact with the binding agent. Conveniently, the binding agents are immobilised at defined, spatially separated locations, to make them easy to manipulate during the assay. The solid support may be a glass surface, e.g. a microscope slide, beads, fibre-optics or microarray chip. In the case of beads, each binding agent may be fixed to an individual bead and they may then be contacted with the expression products in solution.
The sample is generally contacted with the binding agent(s) under appropriate conditions which allow the analyte in the sample to bind to the binding agent(s). The fractional occupancy of the binding sites of the binding agent(s) can then be determined.
Whatever the chosen assay system, there are numerous ways to detect interaction between the binding agent and the expression product (analyte) to be determined, either by directly or indirectly labelling the analyte or binding agent, or by using a developing agent to arrive at an indication of the presence or amount of the analyte in the sample. A developing agent may be a secondary binding agent, capable of binding to a complex between analyte and primary binding agent. For example, if a primary antibody is used as a binding agent, the developing agent may be a secondary antibody capable of binding either to the primary antibody, or to a different epitope on the analyte to that recognised by the primary antibody.
Typically, the analyte, binding agent or developing agent is directly or indirectly labelled (e.g. with radioactive, fluorescent or enzyme labels, such as horseradish peroxidase) so that they can be detected using techniques well known in the art. Directly labelled agents have a label associated with or coupled to the agent. Indirectly labelled agents may act on a further species to produce a detectable result.
Thus, radioactive labels can be detected using a scintillation counter or other radiation counting device, fluorescent labels using a laser, confocal microscope, etc., and enzyme labels by the action of an enzyme label on a substrate, typically to produce a colour change. In further embodiments, the developing agent or analyte is tagged to allow its detection, e.g. linked to a nucleotide sequence which can be amplified in a PCR reaction to detect the analyte. Other labels are known to those skilled in the art are discussed below.
The developing agent(s) can be used in a competitive method in which the developing agent competes with the analyte for occupied binding sites of the binding agent, or non-competitive method, in which the labelled developing agent binds analyte bound by the binding agent or to occupied binding sites. Both methods provide an indication of the number of the binding sites occupied by the analyte, and hence the concentration of the analyte in the sample, e.g. by comparison with standards obtained using samples containing known concentrations of the analyte.
In alternative embodiments, the analyte can be tagged before applying it to the support comprising the binding agent.
There is an increasing tendency in the diagnostic field towards miniaturisation of such assays, e.g. making use of binding agents (such as antibodies or nucleic acid sequences) immobilised in small, discrete locations (microspots) and/or as arrays on solid supports or on diagnostic chips. These approaches can be particularly valuable as they can provide great sensitivity (particularly through the use of fluorescent labelled reagents), require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays can be carried out simultaneously. This latter advantage can be useful as it provides an assay employing a plurality of analytes to be carried out using a single sample. Examples of techniques enabling this miniaturised technology are provided in WO84/01031, WO88/1058, WO89/01157, WO93/8472, WO95/18376/WO95/18377, WO95/24649 and EP 0 373 203 A.
Other methods which do not rely on labelling techniques may also be used to detect interaction between binding agent and reporter molecule, including physical methods such as surface plasmon resonance, agglutination, light scattering or other means.
Expressed nucleic acid (mRNA, pre-mRNA) can be isolated from the cells using standard molecular biological techniques. The expressed nucleic acid sequences corresponding to the gene or genes of Table 1 can then be amplified using nucleic acid primers specific for the expressed sequences in a PCR, e.g. real time PCR, multiplex PCR, etc. The skilled person will be able to select or design a suitable reaction type and protocol depending on, e.g. the number and particular combination of genes to be analysed. If the isolated expressed nucleic acid is mRNA, this can be converted into cDNA for the PCR reaction using standard methods.
The primers may conveniently introduce a label into the amplified nucleic acid so that it may be identified. Ideally, the label is able to indicate the relative quantity or proportion of nucleic acid sequences present after the amplification event, reflecting the relative quantity or proportion present in the original test sample. For example, if the label is fluorescent or radioactive, the intensity of the signal will indicate the relative quantity/proportion or even the absolute quantity, of the expressed sequences. The relative quantities or proportions of the expression products of each of the genes of Table 1 may be used to establish a particular expression profile for the test sample.
Other methods for detection of nucleic acid expression products may also be used, such as in situ hybridisation, Northern blot, etc.
Likewise, protein expression products may be detected by any suitable technique. Immunological techniques are particularly preferred, in which antibodies specific for the particular polypeptide gene product(s), are used as binding agents, although other binding agents such as receptors or ligands capable of binding to the proteins of interest may be employed.
In some embodiments, protein expression products from the sample under test are immobilised on a solid phase and contacted with a binding agent specific for one or more of the proteins of Table 1 under appropriate conditions which allow binding between the protein and the binding agent. The amount of the binding agent found at the surface is then determined. For example, the binding agent may be directly labelled. Alternatively, the immobilised antibody may be contacted with a labelled developing agent capable of binding to the primary antibody. Examples of this type of assay include Western blotting, and certain ELISA (enzyme-linked immunosorbent assay) techniques.
In other embodiments, a binding agent is immobilised on a solid phase and contacted with the sample under suitable conditions to allow binding to take place. The fractional occupancy of the binding sites of the binding agent(s) can then be determined either by directly or indirectly labelling the analyte or by using a developing agent or agents to arrive at an indication of the presence or amount of the analyte in the sample.
An example of this type of assay is an antibody sandwich assay (e.g. an ELISA), which employs two antibodies each capable of binding to a different site on the biomarker protein. The first is immobilised on a solid phase for use as the binding agent. After contact with the analyte, the second antibody is used to detect complexes between the first antibody and analyte.
Whichever method is chosen, it is important that the assay provides a read-out of the level of expression of the biomarker genes which allows results from different individuals to be compared reliably with one another. By way of example, the level of a particular expression product may be determined as a proportion of the total expression products found in the sample. Alternatively, the level of a particular expression product may be determined in relation to the level of expression of a control gene such as a housekeeping gene, or the like. Alternatively, it may be convenient to determine the absolute amount of a particular expression product, e.g. by comparison with known standards. The skilled person will be capable of designing a suitable protocol for any given assay method, and will also be aware of other suitable embodiments.
It has been shown that fragments of a whole antibody can perform the function of binding antigens. The term “antibody” is therefore used herein to encompass any molecule comprising the binding fragment of an antibody, and the term binding agent and binding site should be construed accordingly. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988). In preferred embodiments the binding agent comprises a single antigen binding site specific for the analyte, i.e. a monovalent antibody or antibody fragment.
Pharmaceutical compositions as described in this specification typically comprise, in addition to one or more suitable active agents, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
A group at the University of Ulm, Germany, has recently suggested that a pentapeptide (QYNAD) with Na+ channel-blocking function could be a biological marker of certain inflammatory and immunological disorders of the nervous system16.
The inventors asked whether or not the pentapeptide identified by the German group16 might play a role in CFS. Samples of serum were sent to the University of Ulm for analysis. The 15 samples included 5 normal controls, 5 patients with CFS and 5 disease controls including two patients with MS. Samples were numbered 1-15 and the German group were not informed what the samples were, or which samples were which, until the experiment was concluded.
When the code was broken, the results showed that the disease control group had levels of the pentapeptide which were 2.3× those of the normal controls (similar to the published data) and the CFS samples had levels which were 3× higher than the healthy controls.
Thus, there are measurably higher amounts of the pentapeptide in patients with CFS compared with healthy controls. Although the pentapeptide may not be specific to CFS (as high levels are also found in other disorders), an assay for the peptide could be used as part of the differential diagnosis of CFS.
The German group was unable to identify an endogenous gene which encodes the pentapeptide. The inventors carried out NCBI BLAST and EMBL-Heidelberg Bioccelerator amino acid alignments for the pentapeptide QYNAD. A total of one hundred alignment hits were found. Of these, only nine showed 100% similarity over the five amino acids—five of those were human. The amino acid searches were followed by NCBI BLAST searches using the GenBank Accession and gi numbers for each of the five human amino acid to determine their origins, references and nucleotide sequences. A number of cloned nucleotide sequences were found and when these were run through the nucleotide databases, only one clone showed full-length homology to any human gene. This gene was a human ion-channel gene—the vacuolar proton pump H+-ATPase (v-ATPase). Remarkably, when the human gene amino acid sequence was compared with the original QYNAD pentapeptide it was discovered that the relevant part of the human ion channel encodes the sequence QYMAD.
The inventors next asked whether the v-ATPase represents a candidate gene for a diagnostic test for CFS. RT-PCR using primers specific for the v-ATPase was performed on cDNA prepared from mRNA from PBMCs from CFS patients and healthy controls. As shown in
The vATPase is known to be involved in regulation of a number of metabolic functions which are deranged in CFS/ME. vATPase upregulation could therefore provide an explanation for a number of the symptoms observed. For example, increased vATPase activity could explain the intracellular acidosis in exercising muscles, chest pain (syndrome X), altered neurotransmitter (dopamine) function and abnormal regulation of hypothalamic hormones. In addition, it could explain the increased energy expenditure and fatigue associated with the condition. Taken together, this suggests that the vATPase is not only a marker for the condition, but is a realistic target for intervention therapy.
The inventors went on to examine whether the increase in vATPase expression was confirmed by microarray analysis. Such analysis provides the opportunity to examine the differential expression of mRNA from a very large number of genes. Surprisingly, the results of the analysis not only confirmed their earlier findings regarding the vATPase gene, but also identified differences in the level of expression of key genes in the PBMC of patients with CFS/ME and control subjects, giving an insight into the biochemical pathways which are involved in this disorder.
Microarray results have been verified by western blot analysis and RT-PCR assay. A number of genes, in addition to v-ATPase, were significantly up/downregulated and identified as suitable biomarkers for the disorder.
Advances in genome sequencing and automated chip manufacture have made DNA chip or microarray technology readily available25. This technology allows simultaneous differential expression profiling from a very large number of genes in tissue samples of CFS/ME patients and controls. A recent report from Vernon et al (2002), described a CFS biomarker search in PBMC using a DNA chip array assay which included 1,764 genes. In the study reported here, RNA isolated from PBMC, was assayed using Affymetrix genome-wide chips (HG-U133 arrays) which included 30,000 gene sequences.
Using DNA microarray analysis of whole human genome, gene transcriptional signatures were compared in the PBMC of eight male patients with CFS and seven age-matched male healthy controls. An additional cohort of fourteen patients with CFS and age and sex matched controls was recruited for RT-PCR and western blot assays in order to verify the microarray data. Analysis of the microarray data was performed as described previously (Breitling R. Armengaud P. Amtmann A. Herzyk P. Rank products: A simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Letters 2004; 573(1-3): 83-92; Breitling R. Amtmann A. Herzyk P. Iterative Group Analysis (iGA): A simple tool to enhance sensitivity and facilitate interpretation of microarray experiments. BMC Bioinformatics 2004; 5: (pp 8p)).
Genes which are significantly upregulated in CFS/ME patients compared to healthy controls are detailed in Table 6, ranked according to their RP values. It is considered that any of these genes may be used as biomarkers for CFS/ME. Particularly preferred marker genes are detailed in Table 1.
Further genes, including prostaglandin D2 synthase and T-cell receptors alpha, beta, gamma and delta are found to be down-regulated in CFS patients compared to normal controls. Prostaglandin D2 synthase (NCBI accession no. BC 005939, UniGene Hs.446429) is considered to be a good candidate for a CFS/ME biomarker, because it is known to be involved in sleep regulation; patients with CFS frequently suffer from sleep reversal and fatigue associated with lack of sleep. However, data for other downregulated genes is not shown here. In general, genes which are upregulated in the disease state are considered to be better biomarkers for diagnostic tests etc. than genes which are downregulated because the potential for false-positive tests is significantly higher when using genes which are underexpressed in the disease state.
Iterative group analysis of the differentially expressed genes indicate that in CFS, there is a shift of immune response with preferential antigen presentation to MHC class II receptors and downregulation of T-cell receptor-α, increased cell membrane prostaglandin-endoperoxide synthase activity with downstream changes in oxygen transport and also activation of the guanyl cyclase and caspase pathways of cellular apoptosis. Another set of genes was identified which are involved in the immediate response to infection, particularly by intracellular parasites. The particular genes involved in each of these pathways are identified in Tables 2 to 5. In each of these key pathways, the hub genes were higher ranked in the analysis compared to the network genes.
Functional changes produced by altered gene regulation may explain the mechanism of fatigue and offer a rational basis for targeted pharmacotherapy in CFS.
NCBI accession numbers refer to the UniGene database, build no. 177, released 22 Dec. 2004.
It is clear from this data that significant differences in the expression of a number of genes can be seen in PBMC samples from patients with CFS and healthy controls. We have verified that the DNA microarray assay is valid by confirming the results by RT-PCR and western blot analyses. This is the first time that a reproducible biochemical lesion has been seen in patients with CFS. We propose that bioassays of the significantly over-expressed genes could be used as diagnostic biomarkers for CFS to aid in the differential diagnosis of the condition.
In order to confirm the relevance of the genes identified in the microarray experiments, Western blot and RT-PCR assays have been performed to analyse the expression of selected genes in samples from patients and controls different to those studied in the microarray analysis. The results verify that these genes may be used as potential biomarkers to support the clinical diagnosis of CFS and identify suitable candidates for treatment trials.
Previous reports have hypothesised that CFS is a form of channelopathy—a disorder of membrane ion channels9,11,13. There are several reports in the literature which we believe strengthen the hypothesis that the vacuolar H+ ATPase plays a pathogenic role in CFS.
Local anaesthetics, which are known to act on ion channels, have an adverse effect on patients with CFS/ME. It has been demonstrated also, that in some patients with CFS/ME, there are morphological changes to the red blood cells19. Remarkably, a study by Nishiguchi et al20, has demonstrated that the local anaesthetic lidocaine can induce reversible morphological transformation of human red blood cells and that this change is mediated by the activation of vacuolar H+ ATPase. In addition, Li et al21a have shown that the gene is involved in iron binding in red blood cells.
The ion channel gene is a member of the vacuolar H+ ATPase proton transporting gene family21,22,23. This family of genes is directly involved with the phosphocreatine-dependent glutamate uptake by synaptic vesicles24. The gene is responsible for vesicle docking/exocytosis during neurotransmiter release25 and is a major constituent of synaptic vesicles associated with intracellular membrane structures26. We have demonstrated, using 1H MRS that there is a perturbation of the choline/creatine balance in the CNS (Condon et al17, Chaudhuri et al42). This finding has been corroborated by Puri et al18. As stated above, this type of gene is directly involved in the creatine pathways.
We have previously demonstrated that patients with CFS have low body potassium levels9. Bailey et al27 have shown a relationship between potassium depletion and up-regulation of H+-ATPase.
As stated above, viruses have often been associated with CFS. Virus entry into cells may be mediated by H+ATPase28,29,30. In addition to viral infection affecting neurotransmitter function7, there is a large body of evidence to show that the vacuolar H+-ATPase is also involved31,32,33,34,35,36,37,38,39.
It is clear from the above data that significant differences in the expression of a number of genes can be seen in PBMC samples from patients with CFS and healthy controls. This is the first time that a reproducible biochemical lesion has been seen in patients with CFS. We propose that bioassays of the significantly over-expressed genes (below) could be used as diagnostic biomarkers for CFS to aid in the differential diagnosis of the condition.
Patients with CFS were diagnosed with reference to the 1994 Fukuda definition. All seven patients were male, aged between 18 and 54 years (mean 3 6), and were not on medication. Healthy control subjects were male, aged between 22 and 58 years (mean 34). In addition to the 8 patients and seven control subjects used for the DNA chip assays, an additional fourteen patients and controls were used to confirm the chip assay results by RT-PCR and western blot assays. Informed consent and ethical approval were obtained.
Venous blood samples were drawn from patients who fulfilled the Holmes and Fukuda criteria for ME/CFS, and from healthy individuals. The procedure for isolating PBMC was started immediately and finished within 2 h of sampling. EDTA treated whole blood was diluted 1:1 with phosphate buffered saline. Two volumes of blood were overlaid on one volume of Histopaque −1077 (Sigma Diagnostics) and centrifuged at 20° C. AT 500 g for 30 min. The PBMC interface was removed and washed twice with phosphate buffered saline and centrifuged. The pellets were resuspended in phosphate buffered saline, an aliquot removed and counted in red blood cell lysis buffer (155 Mm NH4CL, 10 Mm NaHCO3, pH7.4, 0.1 mM EDTA). The PBMC were centrifuged once more (20° C., 500 g, 10 min). The PBMC were then aliquoted into microtubes equivalent to 5×105 cells per tube, centrifuged and stored as dry pellets at −80° C.
PBMC pellets were resuspended in sample reducing buffer (1 ml Glycerol, 0.5 ml β-mercaptoethanol, 3 ml 10% SDS, 1.25 ml 1M Tris-HCL Ph 6.7), boiled for 5 min. The lysates were loaded onto a 10% PAGE gel, each track equivalent to 2×10 5 PBMC. The PAGE gel was assessed for equal protein load by coomassie stain. The gel then electrophoretically transferred onto nitrocellulose PVDF membrane (Biorad) for 2 hours. The blots were blocked for non specific binding with 10% normal goat serum for 30 min, probed with a mouse Mab to human defensin1-3, (Hycult, Netherlands), mouse Mab to human thrombospondin (Sigma-Aldrich Inc) and a mouse Mab to Chondroitin Sulphate Proteoglycan (USBiological MA USA), at dilutions of 1/100, 1/1000, 1/1000 respectively in TBS 0.05% Tween 20 for 2 hours at RT. The protein was detected after subsequent incubation with alkaline phosphatase conjugate Goat anti Mouse IgG ( 1/1000 final dilution) (Jackson Immunoresearch Laboratories PA USA). The reactions were detected using SIGMA FAST BCIP/NBT).
Oligonucleotide primers which span a specific epitope within the CFS gene were chosen and tested by RT-PCR. RNA from blood samples from patients with CFS and appropriate controls were RT-PCR amplified and PCR amplicons quantitated by gel documentation system software.
Total RNA was isolated from peripheral blood mononuclear cells (PBMC) using the Promega RNAgents Total RNA Isolation System.
Venous blood was collected in standard EDTA blood tubes and RNA purified using the method of Chomczynski and Sacchi. White blood cell pellets were homogenised by hand in an appropriate volume of denaturing solution (guanidinium thiocyanate, 4M; sodium citrate, 25 mM; N-laurolyl sarcosine, 0.5%; 2-mercaptoethanol, 0.1M, in distilled water adjusted to pH 7.0). To homogenate (1 ml), sodium acetate (100 μl, 2M, pH 4.0), citrate-buffered (0.1M, pH 4.3) phenol (1 ml) and chloroform: isoamyl alcohol (49:1, 200 μl) were sequentially added. The resulting mixture was treated in a vortex mixer (Fisons Scientific Equipment, Whirlimixer™) for 10 seconds then incubated on ice for 15 minutes. Samples were then centrifuged (12,000 g, 20 minutes, 4° C.) and the upper aqueous layer pippetted into to a fresh tube. After addition of ice cold isopropanol (1 ml), RNA precipitated from the mixture during a thirty minute period on dry ice. The mixtures were then centrifuged (12,000 g, 20 minutes, 4° C.) and the supernatant discarded. The pellet was dissolved in denaturing solution (300 μl) and transferred to a microcentrifuge tube (1.5 ml, Axygen). Ethanol (absolute, 600 μl) was added to each microcentrifuge tube, samples were incubated on dry ice for 30 minutes, and then centrifuged (11600 g, 20 minutes, 4° C.). The resulting pellet were then washed twice in ethanol (70% aqueous solution) then lyophylised (using a Hetosicc freeze-drier and a JAVAC high vacuum pump DD-75). The freeze-dried RNA was re-suspended in sterile distilled water (60 μl) and stored at −70° C. until required.
RNA solution (prepared as described above, 2 μl) was added to distilled water (98 μl) to produce a 1:50 dilution. The optical density of the sample was read at 260 nm and 280 nm using a 50 μl ultraviolet cuvet. The absorbance ratio 260/280 nm measured at these wavelengths indicates the purity of the RNA. The absolute concentration of RNA was estimated using the following equation:
Optical Density260nm×40× dilution=RNA concentration (μg/ml)
To examine the quality, 5 μl of RNA was added to 5 μl of the gel marker Orange G (1% Orange G dye in 50% glycerol, 50% 2×TBE). This was heated to 70° C. for 3 minutes, then electrophoresed through a horizontal 1% agarose gel in 1×TBE (10×TBE=0.089M tris(hydroxymethyl)-methylamine, 0.089M boric acid, 0.025M disodium EDTA, pH 8.3). The gel was then stained for 30 minutes in ethidium bromide (0.5 mg/ml) in TBE, de-stained in water and visualised under medium wave (320 nm) ultraviolet light.
cDNA Synthesis
Two micrograms of RNA were added to 1 μl oligo (dT)12-18 (500 μg/ml, Roche) and the volume made up to 11 μl with sterile distilled water. This was incubated (70° C., 10 minutes) to allow the oligo(dT) to bind to the poly-A tail of the RNA, and then chilled on ice. To the reaction mixture, 4 μl of 5× First Strand Buffer, 2 μl of 0.1M DTT, 1 μl of 10 mM dNTP mix (10 mM each dATP, dGTP, dCTP, dTTP; Amersham Pharmacia Biotech), 1 μl of sterile distilled water and 1 μl of Superscript II (Gibco-BRL® Life Technologies) were added. This was incubated first at 50° C. for 1 hr, then 70° C. for 15 minutes to inactivate the reaction. A negative cDNA control (2 μl of distilled water) was included in each cDNA synthesis to confirm that the reaction mix was not contaminated.
To 2 μl of cDNA, 10 μl of 10× magnesium-free buffer, 10 μl of 2.5 mM dNTPs (2.5 mM each dATP, dGTP, dCTP, dTTP), 6 μl MgCl2 (25 mM) and 1 μl each of the appropriate 5′ and 3′ primers (0.5 μg/μl) were added. The volume was made up to 99.8 μl using sterile distilled water and 0.2 μl of Taq DNA polymerase (Promega) was added. The PCR reaction (35 cycles) was carried out on a Techne Genius thermocycler.
Primers used to amplify vATPase were 5′-ctc gtg acc tgt tac tgc tg-3′ and 5′-aag taa cca agt cca ctc ca-3′. Primers for Defensin 1 were 5′-caa gag ctg atg agg ttg ct-3′ and 5′-gaa ggt aca gga gta ata gc-3′.
Thirty microlitres of PCR product was added to 5 μl of orange G and electrophoresed through a horizontal 2-3% agarose gel in 1×TBE at 100 volts, until the dye front had migrated a minimum of 10 cm. On each gel, 3 μl of a 123 base pair DNA ladder in 5 μl of Orange G was included as a size marker for comparison with PCR product bands. The gel was then stained for 30 minutes in ethidium bromide (0.5 mg/ml) in TBE, destained in water and visualised under medium wave (320 nm) ultraviolet light.
Gene expression was analysed by measuring the band density for each amplicon. Band densities were measured using the Herolab EASY Plus computer automated image analysis system. True comparisons in gene expression between samples were enabled by comparing results of densitometry for the experimental genes against the housekeeping gene (abl—tyrosine kinase) band densities.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0502042.5 | Feb 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2006/000332 | 2/1/2006 | WO | 00 | 7/16/2008 |
