WO2011161438A2

WO2011161438A2 - Biomarkers

Info

Publication number: WO2011161438A2
Application number: PCT/GB2011/051145
Authority: WO
Inventors: Sabine Bahn; Emanuel Schwarz
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2010-06-21
Filing date: 2011-06-20
Publication date: 2011-12-29
Anticipated expiration: 2012-12-21
Also published as: WO2011161438A3; GB201010316D0

Abstract

The invention relates to a method of diagnosing or monitoring autism spectrum conditions, in particular Asperger syndrome.

Description

BIOMARKERS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The autism spectrum, also called autism spectrum conditions (ASC) or autism spectrum disorders (ASD), with the word autistic sometimes replacing autism, is a spectrum of psychological conditions characterized by widespread

abnormalities of social interactions and communication, as well as severely restricted interests and highly repetitive behaviour.

Autism spectrum conditions are diagnosed in subjects on the basis of difficulties in communication and social development, including unusually narrow interests, repetitive behaviour and resistance to change (F. R. Volkmar, M . State, A. J. Klin, Child Psychol. Psychiatry. 50, 108 (2009)). Autism spectrum conditions include predominantly classic autism and other forms known as pervasive developmental disorder not otherwise specified (PDD-NOS) and Asperger syndrome (AS). AS differs from classic autism in that subjects are functioning at a higher level with normal to high intelligence quotient (IQ) and do not have a pronounced history of language delay or learning difficulties (M . R. Woodbury-Smith, F.R. Volkmar. Eur. Child Adolesc. Psychiatry. 18, 2 (2009)). The diagnosis of individuals affected by autism spectrum conditions such as AS has risen throughout the past decade and it now appears that more than 1% of the general population is affected . Autism spectrum conditions are thought to be an exaggeration of normal male low-empathizing and high-systemizing

behaviour. Consequently, there is a marked gender difference in the prevalence of these conditions with males being 4-10 times more likely to be affected than females (S. Baron-Cohen et al . Br. J. Psychiatry. 194, 500 (2009); R.C.

Knickmeyer, S. Baron-Cohen S. J. Child Neurol. 21, 825 (2006)). This indicates that sex-linked factors might play a critical role in the aetiology and/or

progression of the condition. In the general population, males tend to perform at lower levels than females on tests of empathy and social sensitivity and they often show stronger abilities at systemizing and visuospatial tasks (S. Baron- Cohen. Trends Cogn. Sci. 6, 248 (2002)). Subjects with autism spectrum conditions show a more pronounced form of these characteristics and this has led to the "extreme male brain" hypothesis as a means of explaining these

conditions at the psychological level (S. Baron-Cohen, (2002), supra). However, little is known about the molecular pathways underlying these effects.

Previous studies have shown that autism spectrum conditions may result from a variety of factors that can affect brain development during gestation. The most widely-reported studies have shown that increased levels of fetal testosterone can lead to differences in brain structure and function, and this has been associated with autism-like traits such as impaired social development and empathy, and increased systemizing behaviour (E. Chapman. Soc. Neurosci. 1, 135 (2006); H.V. Bohm, J. E. Fry McComish, M . G. Stewart. Med. Hypotheses. 69, 47 (2007); E. I. de Bruin et al . Dev. Med. Child Neurol. 48, 962 (2006); B. Auyeung et al. Psychol. Sci. 20, 144 (2009)). This potential link has been supported by the observation of an increased prevalence of testosterone-related disorders in women with autism spectrum conditions (E. Ingudomnukul et al. Horm. Behav. 51, 597 (2007)).

Researchers have also found elevated levels of neurotrophins such as brain- derived neurotrophic factor (BDNF) in archived neonatal blood of children with autism and this has been associated with early brain overgrowth as a potential causative factor (E. Courchesne, R. Carper, N. Akshoomoff. J. A.M. A. 290, 337 (2003)). Another potential pathway has been highlighted by reports of alterations in immune system function and autoimmune phenomena associated with autism spectrum conditions. There are now several studies which have identified an over-activation of mast (M . L. Castellani. Int. J. Immunopathol. Pharmacol. 22, 15 (2009)) and natural killer (A. Vojdani et al. J. Neuroimmunol. 205, 148 (2008)) cells in autistic children and this has been associated with autoimmunity or adverse immune interactions during critical periods of brain development. An increase in prevalence of autism spectrum conditions has also been linked with familial autoimmune diseases and the presence of such disorders as asthma, allergies and psoriasis during pregnancy (A. M. Enstrom, J. A. Van de Water, P. Ashwood. Curr. Opin. Investig. Drugs. 10, 463 (2009),). The interactions of these pathways with the nervous system are likely to be mediated by biological factors, such as hormones and cytokines.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided the use of IL-12p40 as a biomarker for autism spectrum conditions, or predisposition thereto.

According to a second aspect of the invention, there is provided the use of IL- 12p40 optionally in combination with one or more further analytes selected from : Tissue Factor, GOT1, FA BP, IL-3, Erythropoietin, IL-5, G-CSF, IL-1B, Chromogranin A, NrCAM, Tenascin C, TNFalpha, ENA-78, IL-18, Factor VII, CTGF, IL-4, Thrombopoietin, Stem Cell Factor, Sortl, IL-10, IL-12p70 and ICAM-1 as a biomarker for autism spectrum conditions in a male subject, or predisposition thereto. According to a third aspect of the invention, there is provided the use of IL- 12p40 optionally in combination with one or more further analytes selected from : Tissue Factor, GOT1, NARG1, Free Androgen Index, IL-1B, IL-7, BDNF, Apo-CIII, IGM, sRAGE, Apo-Al, Tenascin-C, Eotaxin-3, Endothelin-1, Growth Hormone and insulin or a derivative thereof as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto.

According to a further aspect of the invention, there is provided a method of diagnosing or monitoring autism spectrum conditions, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, one or more of the first analyte biomarkers defined herein.

According to a further aspect of the invention, there is provided a method of diagnosing or monitoring autism spectrum conditions, or predisposition thereto, comprising detecting and/or quantifying, in a sample from a test subject, two or more of the second analyte biomarkers defined herein.

According to a further aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to autism spectrum conditions, comprising detecting and/or quantifying, in a sample from said subject, one or more of the first analyte biomarkers defined herein. According to a further aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to autism spectrum conditions, comprising detecting and/or quantifying, in a sample from said subject, two or more of the second analyte biomarkers defined herein.

A further aspect of the invention provides ligands, such as naturally occurring or chemically synthesised compounds, capable of specific binding to the analyte biomarker. A ligand according to the invention may comprise a peptide, an antibody or a fragment thereof, or an aptamer or oligonucleotide, capable of specific binding to the analyte biomarker. The antibody can be a monoclonal antibody or a fragment thereof capable of specific binding to the analyte biomarker. A ligand according to the invention may be labelled with a detectable marker, such as a luminescent, fluorescent or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g . a biotin, avidin, streptavidin or His (e.g . hexa-His) tag .

A biosensor according to the invention may comprise the analyte biomarker or a structural/shape mimic thereof capable of specific binding to an antibody against the analyte biomarker. Also provided is an array comprising a ligand or mimic as described herein.

Also provided by the invention is the use of one or more ligands as described herein, which may be naturally occurring or chemically synthesised, and is suitably a peptide, antibody or fragment thereof, aptamer or oligonucleotide, or the use of a biosensor of the invention, or an array of the invention, or a kit of the invention to detect and/or quantify the analyte. In these uses, the detection and/or quantification can be performed on a biological sample such as from the group consisting of CSF, whole blood, blood serum, plasma, urine, saliva, or other bodily fluid, breath, e.g . as condensed breath, or an extract or purification therefrom, or dilution thereof.

Diagnostic or monitoring kits are provided for performing methods of the invention. Such kits will suitably comprise a ligand according to the invention, for detection and/or quantification of the analyte biomarker, and/or a biosensor, and/or an array as described herein, optionally together with instructions for use of the kit.

A further aspect of the invention is a kit for monitoring or diagnosing autism spectrum conditions, comprising a biosensor capable of detecting and/or quantifying one or more of the first analyte biomarkers as defined herein.

A further aspect of the invention is a kit for monitoring or diagnosing autism spectrum conditions, comprising a biosensor capable of detecting and/or quantifying two or more of the second analyte biomarkers as defined herein.

Biomarkers for autism spectrum conditions are essential targets for discovery of novel targets and drug molecules that retard or halt progression of the disorder. As the level of the analyte biomarker is indicative of disorder and of drug response, the biomarker is useful for identification of novel therapeutic compounds in in vitro and/or in vivo assays. Biomarkers of the invention can be employed in methods for screening for compounds that modulate the activity of the analyte. Thus, in a further aspect of the invention, there is provided the use of a ligand, as described, which can be a peptide, antibody or fragment thereof or aptamer or oligonucleotide according to the invention; or the use of a biosensor according to the invention, or an array according to the invention; or a kit according to the invention, to identify a substance capable of promoting and/or of suppressing the generation of the biomarker.

Also there is provided a method of identifying a substance capable of promoting or suppressing the generation of the analyte in a subject, comprising administering a test substance to a subject animal and detecting and/or quantifying the level of the analyte biomarker present in a test sample from the subject. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the autism quotient (AQ), empathy quotient (EQ) and systemising quotient (SQ) scale measurements in male and female subjects with Asperger syndrome compared to controls. The interaction p-value was calculated by ANOVA.

Figure 2 provides a Venn diagram showing distinct and common serum biomarker profiles for male and female Asperger syndrome subjects. The arrow indicates the region of markers common to both groups. The analytes highlighted in black text are those which show increased expression and the analytes highlighted in grey text indicate those with decreased expression relative to the values seen in the respective control subjects.

Figure 3 provides a Partial Least Squares Discriminant Analysis showing the separation of Asperger syndrome and control subjects using differentially expressed analytes. The model built on Asperger syndrome males and control males using 24 male specific analytes (left panel) does not yield a separation when predicting Asperger syndrome females and Asperger syndrome controls (right panel).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided the use of IL-12p40 as a biomarker for autism spectrum conditions, or predisposition thereto. Data is presented herein which demonstrates that IL-12p40 was found to demonstrate the most significant fold change in all Asperger syndrome patients when compared with control subjects (see Table 2), IL-12p40 was also found to demonstrate the most significant fold change in male Asperger syndrome patients when compared with control subjects (see Table 3) and IL-12p40 was also found to demonstrate a similar significant level of fold change in female Asperger syndrome patients when compared with control subjects (see Table 4).

In one embodiment, said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1 and IL-1B.

The study presented herein provides evidence of gender specific expression of biomarkers in AS subjects when compared with control subjects. Thus, in one embodiment, said autism spectrum conditions are in a male subject. In an alternative embodiment, said autism spectrum conditions are in a female subject.

In the embodiment wherein the autism spectrum conditions are in a male subject, said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1, IL-1B, FA BP, IL-3, Erythropoietin, IL-5, G-CSF, Chromogranin A, NrCAM, Tenascin C, TNFalpha, ENA-78, IL-18, Factor VII, CTGF, IL-4, Thrombopoietin, Stem Cell Factor, Sortl, IL-10, IL-12p70 and ICAM-1.

In the embodiment wherein the autism spectrum conditions are in a male subject, said one or more analytes are selected from : Tissue Factor, GOT1, FABP, IL-3, Erythropoietin, G-CSF, Chromogranin A, Tenascin C, ENA-78, Factor VII, CTGF, Thrombopoietin, Stem Cell Factor, Sortl, IL-12p70 and ICAM-1.

In the embodiment wherein the autism spectrum conditions are in a male subject, said one or more analytes are selected from : IL-1B, IL-5, NrCAM, TNFalpha, IL-18, IL-4 and IL-10.

In the embodiment wherein the autism spectrum conditions are in a female subject, said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1, IL-1B, NARG1, Free Androgen Index, IL-7, BDNF, Apo-CIII, IGM, sRAGE, Apo-Al, Tenascin-C, Eotaxin-3, Endothelin-1, Growth Hormone and insulin or a derivative thereof. In the embodiment wherein the autism spectrum conditions are in a female subject, said analytes are selected from : Tissue Factor, GOT1, NARG1, Free Androgen Index, IL-7, Apo-CIII, sRAGE, Apo-Al, Tenascin-C, Endothelin-1 and insulin or a derivative thereof. In the embodiment wherein the autism spectrum conditions are in a female subject, said analytes are selected from : IL-1B, BDNF, IGM, Eotaxin-3 and Growth Hormone.

The invention therefore has the advantage of providing gender specific biomarkers for autism spectrum conditions.

The term "biomarker" means a distinctive biological or biologically derived indicator of a process, event, or condition. Analyte biomarkers can be used in methods of diagnosis, e.g . clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment. It will be readily apparent to the skilled person that the analytes listed herein are known and have been described in the literature. References herein to "insulin or a derivative thereof" include references to insulin and derivatives of insulin such as insulin precursors (e.g . proinsulin and des-31,32 proinsulin). According to one particular aspect of the invention which may be mentioned there is provided the use of one or more analytes selected from : IL-12p40, Tissue Factor and GOT1 as a biomarker for autism spectrum conditions, or predisposition thereto. In one embodiment, said analyte additionally comprises IL-1B.

Thus, according to a further particular aspect of the invention which may be mentioned there is provided the use of one or more analytes selected from : IL- 12p40, Tissue Factor, GOT1, IL-1B, FA BP, IL-3, Erythropoietin, IL-5, G-CSF, Chromogranin A, NrCAM, Tenascin C, TNFalpha, ENA-78, IL-18, Factor VII, CTGF, IL-4, Thrombopoietin, Stem Cell Factor, Sortl, IL-10, IL-12p70 and ICAM-1 as a biomarker for autism spectrum conditions in a male subject, or predisposition thereto.

According to a further particular aspect of the invention which may be mentioned there is provided the use of one or more first analytes selected from : IL-12p40, Tissue Factor, GOT1, FABP, IL-3, Erythropoietin, G-CSF, Chromogranin A, Tenascin C, ENA-78, Factor VII, CTGF, Thrombopoietin, Stem Cell Factor, Sortl, IL-12p70 and ICAM-1 as a biomarker for autism spectrum conditions in a male subject, or predisposition thereto.

In one embodiment of any of the aforementioned aspects of the invention, the use additionally comprises one or more second analytes selected from : IL-1B, IL- 5, NrCAM, TNFalpha, IL-18, IL-4 and IL-10.

According to a further particular aspect of the invention which may be mentioned there is provided the use of two or more second analytes selected from : IL-1B, IL-5, NrCAM, TNFalpha, IL-18, IL-4 and IL-10 as a biomarker for autism spectrum conditions in a male subject, or predisposition thereto.

According to a further particular aspect of the invention which may be mentioned there is provided the use of one or more analytes selected from : IL-12p40, Tissue Factor, GOT1, IL-1B, NARG1, Free Androgen Index, IL-7, BDNF, Apo-CIII, IGM, sRAGE, Apo-Al, Tenascin-C, Eotaxin-3, Endothelin-1, Growth Hormone and insulin or a derivative thereof as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto. According to a further particular aspect of the invention which may be mentioned there is provided the use of one or more first analytes selected from : IL-12p40, Tissue Factor, GOT1, NARG1, Free Androgen Index, IL-7, Apo-CIII, sRAGE, Apo- Al, Tenascin-C, Endothelin-1 and insulin or a derivative thereof as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto.

In one embodiment of any of the previously mentioned aspects of the invention, the first analyte is other than Free Androgen Index. In one embodiment of any of the previously mentioned aspects of the invention, the first analyte is other than Apo-Al . In one embodiment of any of the previously mentioned aspects of the invention, the first analyte is other than insulin or a derivative thereof.

Thus, according to a further particular aspect of the invention which may be mentioned there is provided the use of one or more first analytes selected from : IL-12p40, Tissue Factor, GOT1, NARG1, IL-7, Apo-CIII, sRAGE, Tenascin-C and Endothelin-1 as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto.

In one embodiment of any of the aforementioned aspects of the invention, the use additionally comprises one or more second analytes selected from : IL-1B, BDNF, IGM, Eotaxin-3 and Growth Hormone.

According to a further particular aspect of the invention which may be mentioned there is provided the use of two or more second analytes selected from : IL-1B, BDNF, IGM, Eotaxin-3 and Growth Hormone as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto.

In one embodiment of any of the previously mentioned aspects of the invention, the one or more second analytes additionally comprise Free Androgen Index. In one embodiment of any of the previously mentioned aspects of the invention, the one or more second analytes additionally comprise Apo-Al . In one embodiment of any of the previously mentioned aspects of the invention, the one or more second analytes additionally comprise insulin or a derivative thereof. Thus, according to a further particular aspect of the invention which may be mentioned there is provided the use of two or more second analytes selected from : IL-1B, BDNF, IGM, Eotaxin-3, Growth Hormone, Free Androgen Index, Apo-Al and insulin or a derivative thereof as a biomarker for autism spectrum conditions in a female subject, or predisposition thereto.

According to a further particular aspect of the invention which may be mentioned there is provided the use of one or more of the first analytes as hereinbefore defined, as a biomarker for autism spectrum conditions, or predisposition thereto.

According to a further particular aspect of the invention which may be mentioned there is provided the use of two or more of the second analytes as hereinbefore defined, as a biomarker for autism spectrum conditions, or predisposition thereto.

In one embodiment, one or more of the biomarkers may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.

In one embodiment, the autism spectrum condition is selected from autism, Asperger syndrome or pervasive developmental disorder not otherwise specified (PDD-NOS). In a further embodiment, the autism spectrum condition is Asperger syndrome.

According to a further particular aspect of the invention which may be mentioned there is provided the use of one or more analytes as defined hereinbefore as a biomarker for Asperger syndrome, or predisposition thereto. As used herein, the term "biosensor" means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein. Biosensors according to the invention may comprise a ligand or ligands, as described herein, capable of specific binding to the analyte biomarker. Such biosensors are useful in detecting and/or quantifying an analyte of the invention. Diagnostic kits for the diagnosis and monitoring of autism spectrum conditions are described herein. In one embodiment, the kits additionally contain a biosensor capable of detecting and/or quantifying an analyte biomarker.

Monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration and/or remission.

In methods of diagnosing or monitoring according to the invention, detecting and/or quantifying the analyte biomarker in a biological sample from a test subject may be performed on two or more occasions. Comparisons may be made between the level of biomarker in samples taken on two or more occasions. Assessment of any change in the level of the analyte biomarker in samples taken on two or more occasions may be performed. Modulation of the analyte biomarker level is useful as an indicator of the state of the autism spectrum conditions or predisposition thereto. An increase in the level of the biomarker, over time is indicative of onset or progression, i.e. worsening of this disorder, whereas a decrease in the level of the analyte biomarker indicates amelioration or remission of the disorder, or vice versa.

A method of diagnosis or monitoring according to the invention may comprise quantifying the analyte biomarker in a test biological sample from a test subject and comparing the level of the analyte present in said test sample with one or more controls.

The control used in a method of the invention can be one or more control(s) selected from the group consisting of: the level of biomarker analyte found in a normal control sample from a normal subject, a normal biomarker analyte level; a normal biomarker analyte range, the level in a sample from a subject with autism spectrum conditions, or a diagnosed predisposition thereto; autism spectrum conditions biomarker analyte level, or autism spectrum conditions biomarker analyte range.

In one embodiment, there is provided a method of diagnosing autism spectrum conditions, or predisposition thereto, which comprises:

(a) quantifying the amount of the analyte biomarker in a test biological sample; and

(b) comparing the amount of said analyte in said test sample with the amount present in a normal control biological sample from a normal subject.

A higher level of the analyte biomarker in the test sample relative to the level in the normal control is indicative of the presence of autism spectrum conditions, or predisposition thereto; an equivalent or lower level of the analyte in the test sample relative to the normal control is indicative of absence of autism spectrum conditions and/or absence of a predisposition thereto.

The term "diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of autism spectrum conditions, or predisposition thereto. By predisposition it is meant that a subject does not currently present with the disorder, but is liable to be affected by the disorder in time. Methods of monitoring and of diagnosis according to the invention are useful to confirm the existence of a disorder, or predisposition thereto; to monitor development of the disorder by assessing onset and progression, or to assess amelioration or regression of the disorder. Methods of monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development. Efficient diagnosis and monitoring methods provide very powerful "patient solutions" with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), reducing relapse rates. Also provided is a method of monitoring efficacy of a therapy for autism spectrum conditions in a subject having such a disorder, suspected of having such a disorder, or of being predisposed thereto, comprising detecting and/or quantifying the analyte present in a biological sample from said subject. In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control(s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g . prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the level of the biomarker(s) in test samples taken on different occasions.

The invention provides a method for monitoring efficacy of therapy for autism spectrum conditions in a subject, comprising :

(a) quantifying the amount of the analyte biomarker; and

(b) comparing the amount of said analyte in said test sample with the amount present in one or more control(s) and/or one or more previous test sample(s) taken at an earlier time from the same test subject.

A decrease in the level of the analyte biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject is indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder, suspected disorder or predisposition thereto.

Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.

Suitably, the time elapsed between taking samples from a subject undergoing diagnosis or monitoring will be 3 days, 5 days, a week, two weeks, a month, 2 months, 3 months, 6 or 12 months. Samples may be taken prior to and/or during and/or following therapy. Samples can be taken at intervals over the remaining life, or a part thereof, of a subject. The term "detecting" as used herein means confirming the presence of the analyte biomarker present in the sample. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the analyte biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.

In alternative aspects of the invention, the presence of the analyte biomarker is assessed by detecting and/or quantifying antibody or fragments thereof capable of specific binding to the biomarker that are generated by the subject's body in response to the analyte and thus are present in a biological sample from a subject having autism spectrum conditions or a predisposition thereto.

Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of the analyte biomarker in the sample or samples. Biological samples that may be tested in a method of the invention include cerebrospinal fluid (CSF), whole blood, blood serum, plasma, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

Detection and/or quantification of analyte biomarkers may be performed by detection of the analyte biomarker or of a fragment thereof, e.g . a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

The biomarker may be directly detected, e.g . by SELDI or MALDI-TOF. Alternatively, the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.

For example, detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (- TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin- layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used.

Methods of diagnosing or monitoring according to the invention may comprise analysing a sample of cerebrospinal fluid (CSF) by SELDI TOF or MALDI TOF to detect the presence or level of the analyte biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment. Detecting and/or quantifying the analyte biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the analyte biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the analyte biomarkers is performed using two antibodies which recognize different epitopes on a analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtitre plate or strip format.

Immunological methods in accordance with the invention may be based, for example, on any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.

In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the biomarker.

In immunonephelometry, the interaction of an antibody and target antigen on the biomarker results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen, i.e. biomarker, concentration can be determined within minutes of the reaction.

Radioimmunoassay (RIA) methods employ radioactive isotopes such as I¹²⁵ to label either the antigen or antibody. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel . The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.

Enzyme (EIA) immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA). ELISA methods may use two antibodies one of which is specific for the target antigen and the other of which is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemiluminescent or fluorescent signal.

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement. Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.

Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g ., using a sensor chip) or indirect procedure may be used in the detection of analyte biomarkers of the invention.

The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (hapten, antigen, ligand, aptamer, antibody, enzyme etc) is labelled with biotin and the other partner (surface, e.g . well, bead, sensor etc) is labelled with avidin or streptavidin. This is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one. For example a biotinylated ligand (e.g . antibody or aptamer) specific for an analyte biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the analyte biomarker in order to detect and/or quantify an analyte biomarker of the invention. Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.

The term "antibody" as used herein includes, but is not limited to : polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e. g ., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

The identification of key biomarkers specific to a disease is central to integration of diagnostic procedures and therapeutic regimes. Using predictive biomarkers appropriate diagnostic tools such as biosensors can be developed; accordingly, in methods and uses of the invention, detecting and quantifying can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor may incorporate an immunological method for detection of the biomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker(s) at the anticipated concentrations found in biological samples. The biomarker(s) of the invention can be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code" or array configurations. In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker. On exposure, the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram. The test result read-out can be a change in the optical brightness, image, colour and/or position of the image. For qualitative and semi-quantitative applications, a sensor hologram can be read by eye, thus removing the need for detection equipment. A simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor. The format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible. Suitably, biosensors for detection of one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal . Biosensors can be adapted for "alternate site" diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.

Biosensors to detect one or more biomarkers of the invention include acoustic, plasmon resonance, holographic and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the one or more biomarkers of the invention.

Methods involving detection and/or quantification of one or more analyte biomarkers of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g . in the physician's office or at the patient's bedside. Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-neuromedicine. Any suitable animal may be used as a subject non-human animal, for example a non-human primate, horse, cow, pig, goat, sheep, dog, cat, fish, rodent, e.g . guinea pig, rat or mouse; insect (e.g . Drosophila), amphibian (e.g . Xenopus) or C. elegans.

The test substance can be a known chemical or pharmaceutical substance, such as, but not limited to, an anti-ASC disorder therapeutic; or the test substance can be novel synthetic or natural chemical entity, or a combination of two or more of the aforesaid substances.

There is provided a method of identifying a substance capable of promoting or suppressing the generation of the analyte biomarker in a subject, comprising exposing a test cell to a test substance and monitoring the level of the analyte biomarker within said test cell, or secreted by said test cell .

The test cell could be prokaryotic, however a eukaryotic cell will suitably be employed in cell-based testing methods. Suitably, the eukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibian cell (e.g . from Xenopus), C. elegans cell or is a cell of human, non-human primate, equine, bovine, porcine, caprine, ovine, canine, feline, piscine, rodent or murine origin.

In methods for identifying substances of potential therapeutic use, non-human animals or cells can be used that are capable of expressing the analyte.

Screening methods also encompass a method of identifying a ligand capable of binding to the analyte biomarker according to the invention, comprising incubating a test substance in the presence of the analyte biomarker in conditions appropriate for binding, and detecting and/or quantifying binding of the analyte to said test substance.

High-throughput screening technologies based on the biomarker, uses and methods of the invention, e.g . configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g . from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.

Methods of the invention can be performed in array format, e.g . on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.

The invention further provides a substance, e.g . a ligand, identified or identifiable by an identification or screening method or use of the invention. Such substances may be capable of inhibiting, directly or indirectly, the activity of the analyte biomarker, or of suppressing generation of the analyte biomarker. The term "substances" includes substances that do not directly bind the analyte biomarker and directly modulate a function, but instead indirectly modulate a function of the analyte biomarker. Ligands are also included in the term substances; ligands of the invention (e.g . a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, suitably specific binding, to the analyte.

The invention further provides a substance according to the invention for use in the treatment of autism spectrum conditions, or predisposition thereto.

Also provided is the use of a substance according to the invention in the treatment of autism spectrum conditions, or predisposition thereto. Also provided is the use of a substance according to the invention as a medicament. Yet further provided is the use of a substance according to the invention in the manufacture of a medicament for the treatment of autism spectrum conditions, or predisposition thereto. A kit for diagnosing or monitoring autism spectrum conditions, or predisposition thereto is provided . Suitably a kit according to the invention may contain one or more components selected from the group : a ligand specific for the analyte biomarker or a structural/shape mimic of the analyte biomarker, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

The identification of biomarkers for autism spectrum conditions permits integration of diagnostic procedures and therapeutic regimes. Currently there are significant delays in determining effective treatment and hitherto it has not been possible to perform rapid assessment of drug response. Traditionally, many anti-ASC therapies have required treatment trials lasting weeks to months for a given therapeutic approach. Detection of an analyte biomarker of the invention can be used to screen subjects prior to their participation in clinical trials. The biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels. The biomarkers may be used to provide warning of adverse drug response. Biomarkers are useful in development of personalized brain therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions. Thus by monitoring a biomarker of the invention, patient care can be tailored precisely to match the needs determined by the disorder and the pharmacogenomic profile of the patient, the biomarker can thus be used to titrate the optimal dose, predict a positive therapeutic response and identify those patients at high risk of severe side effects. Biomarker-based tests provide a first line assessment of 'new' patients, and provide objective measures for accurate and rapid diagnosis, in a time frame and with precision, not achievable using the current subjective measures. Furthermore, diagnostic biomarker tests are useful to identify family members or patients at high risk of developing autism spectrum conditions. This permits initiation of appropriate therapy, or preventive measures, e.g. managing risk factors. These approaches are recognised to improve outcome and may prevent overt onset of the disorder.

Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder, poor patient compliance or substance abuse. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy or of substance abuse.

The following study illustrates the invention.

This study represents the first reported systematic serum proteome profiling study of adult subjects with Asperger syndrome (hereinafter referred to as AS). In this study, distinct serum biomarker fingerprints for adult male and female subjects with AS were identified using a multiplexed profiling approach. Males showed increased levels of cytokines and other inflammatory molecules. In contrast, female subjects showed altered levels of growth factors and hormones including androgens, growth hormone and insulin-related molecules. This suggests that different compensatory mechanisms occur in males and females or that autism spectrum conditions develop via gender-specific molecular pathways.

Methodology

Patients It was of particular importance to identify any gender-specific differences in the molecular signatures considering the sexually-dimorphic nature of autism spectrum conditions. The protocols for recruitment of the study participants, collection of clinical samples and the test methods were carried out in compliance with the Standards for Reporting of Diagnostic Accuracy (STARD) initiative (P. M . Bossuyt et al. Clin. Chem. 49, 1 (2003)). AS subjects (n=45) were recruited via the Autism Research Centre, the National Autistic Society (UK), Autism social groups and employment services in the UK from 2006-2008. Control subjects (n = 50) were recruited from Cambridge University and the general population using leaflets and advertising over the same time frame and these were matched to AS subjects based on social demographics to ensure no differences in mean age, IQ and body mass index (BMI) (Table 1).

Table 1: Subject information

Age M/F BMI AQ EQ SQ

Control 31.6 + 7.4 26/24 24.0 + 4.1 13.8 + 5.7 47.2 + 15.1 57.8 + 21.7

M 30.1 + 7.9 NA 24.9 + 2.6 18.2 + 5.9 40.2 + 16.1 71 .0 + 25.7

F 33.3 + 6.6 NA 23.7 + 4.5 12.7 + 5.1 49.0 + 14.6 54.2 + 19.6

AS 31.8 + 8.7 22/23 25.7 + 5.1 38.07 + 8.0* 21.7 + 16.5* 79.7 + 24.4

M 30.3 + 8.9 NA 25.3 + 3.5 31 .6 + 7.1* 26.1 + 8.2* 69.4 + 25.0

F 32.9 + 7.7 NA 25.8 + 5.5 40.2 + 7.1* 20.2 + 18.3* 82.9+ 23.8

BMI = body mass index [(weight (kg)/height (m²)]. AQ = autism quotient. EQ = empathy quotient. SQ = systemizing quotient. * p<0.05 in AS subjects compared to controls. The Wechsler Abbreviated Scale of Intelligence (B. N. Axelrod, J. J. Ryan. J. Clin. Psychol. 56, 807 (2000)) was administered to all participants to measure Intelligence Quotient (IQ). Subjects were matched so that there was no difference in age, BMI and IQ. All diagnoses and clinical tests were performed by psychiatrists under Good Clinical Practice (GCP)-compliance to minimize variability. Any patients whose clinical diagnosis required revision at a later stage were excluded from the study. Control subjects with a family history of mental disease or other medical conditions such as type II diabetes, hypertension, cardiovascular or autoimmune diseases were not considered for the study.

AS was diagnosed by psychiatrists based on the Structured Clinical Interview for Diagnostic (SCID) and Statistical Manual IV-Text Revision (DSM-IV-TR). For all subjects, tests were also administered for autism quotient (AQ), empathy quotient (EQ) and systemizing quotient (SQ)-revised (S. Wheelwright et al. Brain Res. 1079, 47 (2006)) to assess the presence and severity of autistic spectrum traits. AS individuals had higher scores for AQ and SQ, and lower scores for EQ, consistent with the diagnosis. Analysis of variance (ANOVA) testing showed a significant interaction between gender and diagnosis for all three scores (AQ, p=0.004; EQ, p=0.014; SQ, p=0.016) with female AS subjects having more extreme symptom scores compared to AS males (see Table 1 and Figure 1).

Assay Methods

147 analytes were measured in 25-50μΙ_ serum samples using the HumanMAP^® multiplexed antigen immunoassays in a CLIA-certified laboratory at Rules Based Medicine (Austin, TX, USA; however, measurement could equally be performed using singleton ELISA). Assays were calibrated using standard curves and raw intensity measurements converted to protein concentrations using proprietary software. Analyses were conducted under blinded conditions with respect to sample identities and the samples were analyzed randomly to avoid any sequential bias due to the presence or absence of disease, patient age or age of material. Analysis Of Variance (ANOVA) was carried out on log transformed data to investigate any interactions between diagnosis and gender for each molecular analyte and psychometric characteristic (AQ, EQ and SQ). The data were also assessed for deviations of normality and homoscedasticity using the Aligned Rank Transform (ART) non-parametric analyses to test for interactions.

Results

Analysis of analytes altered in AS subjects using Human MAP®

HumanMAP® profiling of 147 analytes was conducted on serum samples from the AS and control subjects in a Clinical Laboratory Improved Amendments (CLIA)-certified laboratory. Twenty-five analytes were identified that showed significant differences in expression between AS and control subjects (Table 2).

Table 2: Analytes altered in all AS subjects p-value p-value

Analyte diagnosis gender Interaction* FC

IL-12p40 <0.001 0.219 0.843 2.83

NARG1 0.014 0.367 0.1 12 1.90

IL-1 B 0.001 0.084 0.324 1.58

Tissue factor 0.001 0.028 0.893 1.56

Leptin 0.018 <0.001 0.789 1.44

IL-5 0.002 0.018 0.099 1.37

Luteinizing hormone 0.047 0.018 0.244 1.34

IL-7 0.001 0.076 0.059 1.27

Alpha fetoprotein 0.028 0.943 0.453 1.26 ENA-78 0.029 0.001 0.701 1.22

IL-18 0.042 0.302 0.254 1.17

Factor VII 0.015 0.508 0.358 1.15

BDNF 0.012 0.221 0.127 1.12

IL-15 0.020 <0.001 0.841 1.10

Thrombopoietin 0.012 0.001 0.022 1.09

Thyroxine binding globulin 0.021 0.027 0.753 1.09

Sortl 0.040 0.920 0.144 1.08

Cortisol 0.047 <0.001 0.226 1.08

GOT1 0.001 <0.001 0.882 0.84

IgA 0.028 <0.001 0.278 0.87

Apo-A1 0.006 0.001 0.054 0.82

igM 0.020 <0.001 0.398 0.81

Ferritin 0.009 <0.001 0.580 0.70

Endothelin-1 0.001 0.033 0.034 0.59

Angiotensinogen 0.034 0.488 0.760 0.57

Tenascin C 0.488 0.193 <0.001

MMP-9 0.093 0.773 0.002

Stem cell factor 0.413 0.007 0.005

FABP 0.099 0.677 0.005

FGF basic 0.297 0.227 0.007

CTGF 0.864 0.754 0.009

Chromogranin A 0.072 0.362 0.011

Growth hormone 0.072 <0.001 0.021

TNFa 0.061 0.716 0.022

Free androgen index 0.473 <0.001 0.022

sRAGE 0.945 0.255 0.024

Complement 3 0.983 0.051 0.031

GM-CSF 0.367 0.002 0.047

SHBG 0.812 <0.001 0.050

Two-way ANOVA for effects of diagnosis (difference between AS and controls) and gender (difference between males and females).

*Two-way ANOVA interaction of diagnosis and gender.

FC = fold change. Average intensity of analyte in AS divided by the average intensity in NC.

Free androgen index = total testosterone concentration divided by SHBG concentration.

The upper portion of the table shows the analytes that have a significant diagnosis main effect. The lower portion includes all other analytes that featured a significant diagnosis-gender interaction. Marked changes were observed for IL-12p40, NMDA receptor regulated 1 (NARG1), erythropoietin (EPO), tissue factor, IL-1B and endothelin-1, which were altered by more than 1.5-fold. ANOVA testing found that 15 of these analytes also showed differences in expression between males and females. In addition, 14 separate analytes showed significant interactions between diagnosis and gender. Total testosterone showed no difference between Asperger syndrome subjects and controls and there was no significant diagnosis-gender interaction. In contrast, the free testosterone levels showed a significant diagnosis-gender interaction as determined by calculating the free androgen index (FAI), which is defined as the ratio of total testosterone to the sex hormone binding globulin (SHBG). Without being bound by theory, it is likely that such gender-related molecular differences may obscure any molecular signatures associated with AS when samples from males and females are analyzed together. For this reason, male and female samples were analyzed separately in the remaining studies. This approach resulted in identification of 24 analytes that were altered significantly in AS males (Table 3) and 16 that were altered significantly in AS females (Table 4) . Table 3: Analytes altered in Male AS subjects

Analvte p-value FC

IL-12p40 0.0015 2.88

FABP 0.0010 2.02

IL-3 0.0313 1.98

Erythropoietin 0.0093 1.92

Tissue Factor 0.0001 1.70

IL-5 0.001 1 1.59

G-CSF 0.0042 1.47

IL-1 B 0.0034 1.44

Chromogranin A 0.0124 1.42

NrCAM 0.0303 1.36

Tenascin-C 0.0300 1.32

TNFa 0.0044 1.29

ENA-78 0.0493 1.26

IL-18 0.0186 1.24

Factor VII 0.0316 1.21

CTGF 0.0090 1.19

IL-4 0.0267 1.19

Thrombopoietin 0.0022 1.19

Stem cell factor 0.0098 1.18

Sortl 0.0078 1.14

IL-10 0.0333 1.12

IL-12p70 0.0466 1.10

ICAM-1 0.0466 1.10

GOT1 0.0008 0.83

F/M p-value = significant difference between control females and control males (gender interaction)

Table 4: Analytes altered in Female AS subjects

Analvte p-value FC

NARG1 0.0082 2.87

IL-12p40 0.001 1 2.74

Free androgen index 0.0001 2.01

IL-1 B 0.0073 1.75

IL-7 0.0224 1.47

Tissue Factor 0.0328 1.42

BDNF 0.0150 1.20

GOT1 0.238 0.84

Apo-CIII 0.0438 0.83 IGM 0.0130 0.76

sRAGE 0.0425 0.76

Apo-A1 0.0063 0.75

Tenascin-C 0.0021 0.68

Eotaxin-3 0.0266 0.53

Endothelin-1 0.0369 0.50

Growth hormone 0.0103 0.44

F/M p-value = significant difference between control females and control males (gender interaction) Free androgen index = testosterone/SHBG.

Only 4 analytes (IL-12p40, tissue factor, IL-1B, GOT1) were changed in the same direction in males and females, providing evidence for a minimal overlap of the molecular signatures (Figure 2). One protein (tenascin-C) showed the opposite regulation as this was increased in males and decreased in females.

Multivariate statistical classification using partial least squares descriminant analysis (PLS-DA) revealed that the combined panel of 24 differentially expressed analytes in males produced a separation between AS and control subjects with a sensitivity of 0.86 and a specificity of 0.88 (Figure 3). However, testing this same panel of 24 analytes in the female group did not result in a separation between AS and controls (Figure 3). This lent further support to the case that the underlying molecular signature is distinct between male and female AS subjects. Analytes that were altered specifically in males included several cytokines (IL-3, IL-4, IL-5, IL-10, IL-12p70, TNFa, ENA-78), fatty acid binding protein (FABP), the neuroendocrine secreted protein chromogranin A, and the cardiovascular and blood cell-associated proteins thrombopoietin (TPO) and erythropoietin (EPO). The finding that the unique fingerprint for male AS subjects was comprised mostly of increased levels of cytokines is consistent with previous reports suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of autism spectrum conditions (Vojdani et al (2008) supra; Enstrom et al (2009) supra; Bossuyt et al (2003) supra). The female-specific AS analytes showed a marked difference in nature and included growth factors and hormones such as growth hormone, endothelin-1, brain derived neurotrophic factor (BDNF), luteinizing hormone and free testosterone. This is consistent with previous reports on alterations of growth factors, peptidyl and steroidal hormones in these conditions (Chapman (2006) supra; Bohm (2007) supra; de Bruin et al (2006) supra; Auyeung et al (2009) supra; Ingudomnukul et al (2007) supra; Courchesne et al (2003) supra; Castellani (2009) supra).

This is the first study showing that free testosterone is increased in AS and that this effect was specific for females affected by this condition. The elevation of androgens in children with autism spectrum conditions has been observed previously and has led to the suggestion of employing androgen-lowering therapies for managing some of the associated symptoms (D. A. Geier, M . R. Geier. Neuro. Endocrinol. Lett. 28, 565 (2007)). The increased testosterone levels in AS females was consistent with the finding of increased levels of luteinizing hormone (LH) in the same subjects. This is interesting as previous studies have shown that LH pulsatility may predispose or cause hyperandrogenism in some female adolescents (M . E. Escobar et al. Horm. Res. 68, 278 (2007)) and has been associated with such disorders as polycystic ovarian syndrome (PCOS) (M . Barontini, M . C. Garcia-Rudaz, J. D. Veldhuis. Arch. Med. Res. 32, 544 (2001)). BNDF was also increased specifically in AS females, consistent with previous studies showing increased levels of this growth factor in children with autism spectrum conditions in association with abnormal cerebral development (Castellani (2009) supra).

Female AS subjects also showed an approximate 2-fold decrease in growth hormone levels, supporting previous studies which found a growth hormone deficit in autism (L. Ragusa, M . Elia, R. Scifo. Growth hormone deficit in autism. J. Autism Dev. Disord. 23, 421). This effect may be linked to perturbed hypothalamic pituitary adrenal (HPA) axis function which has been reported in individuals with autism spectrum conditions in response to an insulin challenge (K. R. Maher et al . J.Nerv. Ment. Dis. 161, 180 (1975)). Therefore, we looked at the levels of insulin in AS compared to controls. There was no change for male AS subjects (p=0.469) although AS females showed a non-significant 1.57-fold increase compared to levels observed in control females (p=0.208) (data not shown). As insulin is co-released with residual levels of unprocessed proinsulin and the des31,32-proinsulin conversion intermediate, we also measured the levels these molecules specifically using sensitive two-site time resolved fluorometric (TRF) assays. For insulin, the results of TRF assay showed a high correlation (r²=0.98) with the multiplexed xMAP^® platform with an increase in AS females of 1.81-fold compared to control females although, again, this did not reach significance (Table 5). Table 5: Two-site TRF assays for insulin-related molecules in female AS subjects

Analyte p-value FC

Insulin 0.0594 1.81

Proinsulin 0.2508 1.26

31.32-PI 0.0228 1.89

Glucose levels showed a trend increase in AS females compared to control females (FC=1.11; p=0.0670). Glucose was determined spectrophotometrically using an adaptation of the hexokinase- glucose-6-phosphate dehydrogenase method in a Dimension RXL Clinical Chemistry System (Dade Behring; Milton Keynes, UK). Insulin was measured using a two-step time resolved fluorometric (TRF) assay from Perkin Elmer (Beaconsfield, Bucks, UK). Proinsulin and des31 ,32-proinsulin (31 ,32-PI) were determined using two-site TRF assays employing combinations of monoclonal antibodies which can distinguish between the proinsulin forms. The levels of proinsulin were not altered significantly although des31,32- proinsulin showed a significant 1.89-fold increase in females. The finding that glucose levels were relatively normal in the same subjects (1.11-fold; p=0.0670) suggested that the relatively high levels of insulin-related molecules may be due to insulin resistance. This is intriguing because of the link between hyperinsulinemia and hyperandrogenism as described in previous studies of PCOS and post-menopausal women (L. Kebapcilar et al. Arch. Gynecol. Obstet. Mar 28 (2009) [EPub] ; S. H . Golden et al. Am. J. Epidemiol. 160, 540 (2004)). Importantly, treatment with insulin sensitizing agents such as rosiglitazone has been shown to improve insulin sensitivity, leading to alleviation of hyperandrogenism and the associated symptoms (Z. Zheng et al. Zhonghua Fu Chan Ke Za Zhi. 37, 271 (2002)).

This study has demonstrated for the first time that males and females with AS have distinct serum molecular signatures. AS males featured a higher number of altered analytes which were increased almost exclusively and showed only a small overlap with molecules changed in AS females. This provides evidence that the peripheral molecular signature of AS is different in male and female subjects and suggests that interpretation of past studies of autism spectrum conditions may have been hampered by not considering male and female subjects separately. In particular, males showed increased levels of cytokines and other inflammatory molecules, consistent with the proposed role of these pathways in autism spectrum disorders (T. van Gent, C. J. Heijnen, P. D. Treffers. J. Child Psychol. Psychiatry. 38, 337 (1997) ; C. A. Molloy et al. J. Neuroimmunol. 172, 198 (2006)). However, female subjects showed a distinct pattern with alterations in the levels of various growth factors and hormones including androgens, growth hormone and insulin-related molecules. This could explain the increased "maleness" observed in females with autism spectrum conditions. These gender- distinct molecular profiles suggest that either different compensatory mechanisms occur in males and females with autism spectrum conditions or that these conditions may develop through distinct gender-specific molecular pathways.

Claims

1. Use of IL-12p40 as a biomarker for autism spectrum conditions, or predisposition thereto.

2. Use as defined in claim 1, wherein said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1 and IL-1B.

3. Use as defined in claim 1, wherein said autism spectrum conditions are in a male subject.

4. Use as defined in claim 3, wherein said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1, IL-1B, FABP, IL-3, Erythropoietin, IL-5, G-CSF, Chromogranin A, NrCAM, Tenascin C, TNFalpha, ENA-78, IL-18, Factor VII, CTGF, IL-4, Thrombopoietin, Stem Cell Factor, Sortl, IL-10, IL-12p70 and ICAM-1.

5. Use as defined in claim 4, wherein said one or more analytes are selected from : Tissue Factor, GOT1, FABP, IL-3, Erythropoietin, G-CSF, Chromogranin A, Tenascin C, ENA-78, Factor VII, CTGF, Thrombopoietin, Stem Cell Factor, Sortl, IL-12p70 and ICAM-1.

6. Use as defined in claim 4, wherein said one or more analytes are selected from : IL-1B, IL-5, NrCAM, TNFalpha, IL-18, IL-4 and IL-10.

7. Use as defined in claim 1, wherein said autism spectrum conditions are in a female subject.

8. Use as defined in claim 7, wherein said use additionally comprises one or more analytes selected from : Tissue Factor, GOT1, IL-1B, NARG1, Free

Androgen Index, IL-7, BDNF, Apo-CIII, IGM, sRAGE, Apo-Al, Tenascin-C, Eotaxin-3, Endothelin-1, Growth Hormone and insulin or a derivative thereof.

9. Use as defined in claim 8, wherein said analytes are selected from : Tissue Factor, GOT1, NARG1, Free Androgen Index, IL-7, Apo-CIII, sRAGE, Apo-Al, Tenascin-C, Endothelin-1 and insulin or a derivative thereof.

10. Use as defined in claim 8, wherein said analytes are selected from : IL-1B, BDNF, IGM, Eotaxin-3 and Growth Hormone.

11. A method of diagnosing or monitoring autism spectrum conditions, or predisposition thereto comprising detecting and/or quantifying, in a sample from a test subject, the analyte biomarkers as defined in any of claims 1 to 10.

12. A method of monitoring efficacy of a therapy in a subject having, suspected of having, or of being predisposed to autism spectrum conditions, comprising detecting and/or quantifying, in a sample from said subject, the analyte biomarkers as defined in any of claims 1 to 10.

13. A method as defined in claim 11 or 12, which is conducted on samples taken on two or more occasions from a test subject.

14. A method as defined in any of claims 11 to 13, further comprising comparing the level of the biomarker present in samples taken on two or more occasions.

15. A method as defined in any of claims 11 to 14, comprising comparing the amount of the biomarker in said test sample with the amount present in one or more samples taken from said subject prior to commencement of therapy, and/or one or more samples taken from said subject at an earlier stage of therapy.

16. A method as defined in any of claims 11 to 15, further comprising detecting a change in the amount of the biomarker in samples taken on two or more occasions.

17. A method as defined in any of claims 11 to 16, comprising comparing the amount of the biomarker present in said test sample with one or more controls.

18. A method as defined in claim 17, comprising comparing the amount of the biomarker in a test sample with the amount of the biomarker present in a sample from a normal subject.

19. A method as defined in any of claims 11 to 18, wherein samples are taken prior to and/or during and/or following therapy for autism spectrum conditions.

20. A method as defined in any of claims 11 to 19, wherein samples are taken at intervals over the remaining life, or a part thereof, of a subject.

21. A method as defined in any of claims 11 to 20, wherein quantifying is performed by measuring the concentration of the analyte biomarker in the or each sample.

22. A method as defined in any of claims 11 to 21, wherein detecting and/or quantifying is performed by one or more methods selected from SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC or other LC or LC-MS-based technique.

23. A method as defined in any of claims 11 to 22, wherein detecting and/or quantifying is performed using an immunological method.

24. A method as defined in any of claims 11 to 23, wherein the detecting and/or quantifying is performed using a biosensor or a microanalytical, microengineered, microseparation or immunochromatography system.

25. A method as defined in any of claims 11 to 24, wherein the biological sample is cerebrospinal fluid, whole blood, blood serum, plasma, urine, saliva, or other bodily fluid, or breath, condensed breath, or an extract or purification therefrom, or dilution thereof.

26. A kit for monitoring or diagnosing autism spectrum conditions, comprising a biosensor capable of detecting and/or quantifying the analyte biomarkers as defined in any of claims 1 to 10.

27. A use, method or kit as defined in any preceding claims wherein said autism spectrum condition is Asperger syndrome.