WO2011026185A1 - An assay for monitoring a neurological condition - Google Patents

Abstract

The present invention relates generally to biomarkers of neurological conditions including monitoring disease progression and the clinical severity of symptoms. More particularly, the present invention provides an assay to facilitate diagnosis and monitoring of progression of neurological conditions and treatment response and to identify targets for therapeutic intervention. Kits and assays for diagnosis and identification of medicaments also form part of the present invention.

Description

AN ASSAY FOR MONITORING A NEUROLOGICAL

CONDITION

FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2009904229, filed on 4 September 2009, entitled "A method of treatment and disease monitoring", the entire contents of which, are incorporated herein by reference.

FIELD

[0002] The present invention relates generally to biomarkers of neurological conditions including monitoring disease progression and the clinical severity of symptoms. More particularly, the present invention provides an assay to facilitate diagnosis and monitoring of progression of neurological conditions and treatment response and to identify targets for therapeutic intervention. Kits and assays for diagnosis and identification of medicaments also form part of the present invention. BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description. [0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Parkinson's disease (PD) is a debilitating and prolonged condition. PD is in fact a spectrum of neurological conditions with clinically undetectable symptoms to a condition characterized by severe motor and nonmotor handicap (Louis and Luchsigner, Arch Neurol 63:717-722, 2006; Richards et al, Neurology ¥3:2184-2188, 1993; Wilson et al, Neurology 55: 1815-1819, 2002; Bennet et al, N. Engl. J. Med. 334:71 -76, 1996; Yamouchi and Nagura, Stroke 25:965-969, 1997).

[0006] Complex pathways appear to contribute to the symptoms and signs of PD. The currently recognized genetic forms of PD account for around 1 % of cases. There is a need to identify critical mutations which exacerbate PD and its related conditions and which influence any response to treatment.

[0007] The Fragile X Mental Retardation genetic locus ("FMR genetic locus") includes the FMR1 gene which is composed of 17 exons, spanning 38Kb, and encodes Fragile X Mental Retardation Protein (FMRP), essential for normal neurodevelopment (Verkerk et al, Cell 65(5) :9QS-9 \ A, 1991 ; Terracciano et al, Am JMed Genet C Semin Med Genet 137C(l):32-37 , 2005). A CGG repeat segment is located within the 5' untranslated region (UTR) of the gene. Its normal range is between 5 to 34 repeats. When expanded, these repeats have been implicated in a number of pathologies, including the Fragile X syndrome (FXS), Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and premature ovarian failure (POF). FXS is neurodevelopmental in nature with a frequency of 1/4000 males and 1/8000 females, associated with a Fragile site at the Xq27.3 locus (Jin and Warren, Hum. Mol. Genet 9(6):90l-908, 2000).

[0008] FXS is caused by CGG expansion to "full mutation" (FM) which comprises >200 repeats, leading to a gross deficit of FMRP and subsequent synaptic abnormalities (Pieretti et al, Cell 66(4): \ l- 22, 1991 ; Irwin et al, Cereb Cortex 70( 0j: l O38- l O44, 2000). The FXS clinical phenotype ranges from learning disabilities to severe mental retardation and can be accompanied by a variety of physical and behavioral characteristics. FXTAS is prevalent in -30% of premutation individuals (PM), comprising - 55 to 199 repeats (Nolin et al, Am JHum Genet 72^:454-464, 2003) and is a progressive neurodegenerative late-onset disorder with a frequency of 1/3000 males in the general population (Jacquemont et al, Am J Ment Retard 109(2) \ 54- \ 64, 2004), manifesting as tremor, imbalance and distinct MRI and histological changes (Hagerman et al, Neurology 57(1): 127- 130, 2001 ; Jacquemont et al, JMed Genet 42(2):e \ 4, 2005; Loesch et al, Clin Genet 67 (5):4 \ 2-4 \ l ^', 2005). It is often associated with 'toxicity' of elevated FMRl mRNA, which has been linked to the intranuclear inclusions and cell death observed during neurodegeneration (Jin et al, Neuron 39(5) :Ί 9-Ί4Ί , 2003).

[0009] There is a need to identify links between expression within the FMR genetic locus and the dysfunction of mitochondrial pathways and/or the dopaminergic system. Abnormal function of the dopaminergic system contributes to pathologies within the spectrum of Parkinsonism, and major neuropsychiatric conditions including Alzheimer's Disease (Proitsi et al, Neurobiol Aging, 2010), schizophrenia (van Os and Kapur, Lancet 22 374(9690):635-45, 2009), bipolar disorder (Cousins et al, Biopolar Disord (¾⁾:787-806, 2009), chronic depression (Monte-Silva et al, J Psychol, 2010) and autism (Baskerville and Douglas, CNS Neurosci Ther. (3 :e92-123, 2010; van Os and Kapur, 2009 supra).

VERIFICATION

[0010] The present case claims priority from Australian Provisional Application No. 2009904229, filed 4 September, 2009. In the provisional specification, it was observed that expression of Cytochrome C subunit 1 (CYC1) was significantly elevated in blood of GZ and low PM patients, and this was shown to be associated with UPDRS amongst other clinical measures of the clinical phenotype, suggesting that mitochondrial dysfunction in these carriers is linked to the clinical outcome. CYC1 is a nuclear oxidative- phosphorylation gene expressed during periods of mitochondrial expansion (Li et al, Eur J Biochem 241 (2): 649-656, 1996). The findings reported in the provisional specification indicating mitochondrial dysfunction in carriers of GZ and PM expansions with Parkinsonism has since been supported by Ross-Inta et al, Biochem J 429:545-552, 2010. Furthermore, an aspect of the present invention relating to the association of Parkinsonism and smaller FMRl expansions has also been supported by Hall et al, Parkinsonism and Related Disorders 2010, doi: 10.1016/j.parkreldis.2010.07.006. [0011] Furthermore, the provisional specification outlined that FMR1 mRNA and FMRP levels were decreased in lymphoblasts of patients with pathologies within the spectrum of Parkinsonism, untreated with levodopa or its analogs and with normal size CGG expansions. It was further observed that, in patients matched for age, sex and disease duration, but treated with levodopa, and with normal size CGG expansions, the levels of FMRP in lymphoblasts were equivalent to healthy controls matched for age and sex. The treated patients were provided with levodopa to alleviate the symptoms by normalizing defects in their dopaminergic system contributing to the severity of the disease. Because dopamine supplementation was accompanied with increase in FMR1 mRNA and FMRP levels, it indicated that mRNA and protein expression within the FMR genetic locus may be regulated by the dopaminergic system and this may be related to neuropsychiatric disorders including, but not restricted to dementia (Proitsi et al, 2010 supra), schizophrenia (van Os and apur, 2009 supra), bipolar disorder (Cousins et al, 2009 supra), chronic depression (Monte-Silva et al, 2010 supra) and autism spectrum disorders (Baskerville and Douglas,2010 supra; van Os and Kapur, 2009 supra). These findings of decreased FMR1 mRNA and FMRP expression in neuropsychiatric disorders suggesting a link to dysfunction in the dopaminergic system have since been supported by Fatemi et al, Schizophr. Res. 2010, doi: 10.1016/j.schres.2010.07.017).

SUMMARY

[0012] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0013] The present invention identifies biomarkers of neurological disease conditions including their clinical severity and their progression prior to or during treatment. The biomarkers include the level of FMR1 gene expression within the FMR genetic locus, the level of expression of mitochondrial and/or apoptotic genes such as cytochrome C I (CYC1) and Caspase 8 or the genes encoding or associated with mitochondrial complex III. The levels of expression of these biomarkers especially in the context of the presence, absence and/or extent of CGG expansion is instructive as the presence or severity of a neurological condition and/or its progression prior to or during treatment. It is demonstrated herein that the size of the CGG repeat expansion, within both GZ and lower PM range, is a significant factor contributing to the origin and manifestations of the neurological disease condition. Furthermore, toxicity of FMR1 sense and ASFMR1 antisense transcripts in both these allele categories is linked to mitochondrial dysfunction and dopaminergic system dysfunction. Reference to "expression" in relation to a particular biomarker includes level of gene expression as well as level of protein produced.

Reference to a "neurological disease or condition" includes a neurodegenerative disorder as well as a disease or condition having symptoms of a pathology or disorder involving the mitochondrial and/or dysregulation of the dopaminergic system, including but not restricted to a disease within the Parkinsonism spectrum, dementia, schizophrenia, bipolar disorder, chronic depression and autism spectrum disorders. A "neurological condition" includes a neuropathological condition. The term "Parkinsonism" includes typical and atypical Parkinson's disease (PD), as well as Parkinsonian syndromes such as Parkinsonism spectrum conditions and Parkinson's disease spectrum (PDS). The terms "disease" and "condition" may be used interchangeably throughout this specification. A neurological condition may exist which is not clinically classified as a disease. Such conditions include behavioral characteristics, memory and cognitive levels.

[0014] Hence, one aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of a genetic region or protein selected from FMR1 , ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject, wherein clinical symptoms are considered severe when: (i) there is an elevation in expression of FMR1 and/or ASFMR1 in whole blood and its constituents or a decrease in expression of FMR1 in Epstein Barr virus (EBV)- transformed lymphocytes in the presence of a CGG expansion associated with a grey zone (GZ) mutation compared to healthy controls; (ii) there is a decrease expression of FMR1 mRNA and/or protein in EBV-transformed lymphocytes in the absence of any CGG expansion compared to healthy controls;

(iii) there is an increase in expression of CYC1 or Caspase 8 in the presence of a GZ expansion compared to healthy controls;

(iv) there is a decrease in expression of CYC1 in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls;

(v) there is an increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls; and/or

(vi) there is an increase in expression of FMR1 sense and ASFMR1 antisense transcripts.

[0015] Detection of dysfunction of the dopaminergic pathology is also an indication of enhanced clinical severity with respect to the neurological condition.

[0016] Another aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of a genetic region selected from FMRl , ASFMRl , CYCl and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein clinical symptoms are considered severe when there is an elevation in expression of FMRl in whole blood and its constituents or a decrease in expression of FMRl and/or ASFMRl and/or its product FMRP in Epstein Barr virus (EBV)-transformed lymphocytes or other blood constituents in the presence of a CGG expansion associated with a grey zone (GZ) mutation compared to patients with a neurological condition with normal size CGG expansion in FMRl .

[0017] The assay of the present invention is useful for monitoring treatment of a subject with a neurological condition. In addition, the degree of severity of the neurological condition, such as but not limited to Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and autism spetrum disorders is linked to the level of FMRl and ASFMRl expression and FMRP. The greater the expression of FMRl and/ASFMRl mRNA, the more severe the condition. The lower the levels of FMRP, the more severe the condition. As indicated above, Parkinsonism includes Parkinson's disease spectrum, Parkinsonian syndromes, and typical and atypical Parkinson's disease.

[0018] The present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of a genetic region or protein selected from FMRl, ASFMRl, CYCl and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein the treatment protocol is considered effective when: (i) there is a decrease in expression of FMRl or ASFMRl in whole blood and its constituents or a decrease in expression of FMRl mRNA and/or FMRP in EBV- transformed lymphocytes in the presence of a GZ mutation;

(ii) there is an increase or decrease in expression of FMRl and/or FMRP in EBV- transformed lymphocytes in the absence of any CGG expansion;

(iii) there is a decrease in expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) there is an increase in expression of CYCl and Caspase 8 and their protein products in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is a decrease in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls;

(vi) there is an increase in expression of mitochondrial complex III in EBV-transformed lymphocytes in the absence of a GZ expansion; and/or

(vii) there is a decrease or normalization in expression of FMRl and ASFMRl .

[0019] The present invention further provides for the use of expression of a gene or protein selected from FMRl, ASFMRl , CYCl and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in the manufacture of an assay to determine the clinical severity of a neurological condition or the efficacy of a therapeutic protocol to a neurological condition.

[0020] In one embodiment, the neurological condition is Parkinsonism. This includes typical and atypical PD, Parkinsonian syndromes and Parkinson's disease spectrum. The present invention further establishes that the effect of CGG expansions on neurological phenotype, including Parkinsonism, is mediated in part through mitochondrial or dopaminergic dysfunction. This means that neurological conditions can be further managed by screening for mitochondrial or dopaminergic dysfunction. These also provide other targets for therapeutic intervention. In another embodiment, the neurological condition is dementia, schizophrenia, bipolar disorder, chronic depression or autism spectrum disorder.

[0021] Kits for conducting the assays of the present invention as well as assays for screening for medicaments for use in ameliorating symptoms of neurological conditions also form part of the present invention. [0022] A list of abbreviations used herein is provided in Table 1.

Table 1

Abbreviations

Abbreviation Definition

ASFMR1 Antisense FMRl (effectively equivalent to FMR4)

(CGG)n CGG repeat element located within 5' untranslated region of the FMRl gene

CYC1 Cytochrome C 1

DNMT DNA-methyltransferase

EBV Epstein Barr Virus

FM Full mutation (>200 CGG repates)

FMR genetic Fragile X mental retardation genetic locus

locus

FMRl Fragile X mental retardation Gene 1

FMR4 Fragile X mental retardation Gene 4 (effectively equivalent to

ASFMR1)

FMRP Protein encoded by FMRl

GZ Grey zone expansion with the 5'UTR of the FMRl gene (40-55 CGG repeats)

ICARS International Cooperative Ataxia Rating Scale

L-dopa L-3,4-dihydroxyphenylalanine; Levodopa

MLPA Multiplex ligation-dependent probe amplification

MMS Mini-Mental State

MMSE Mini-Mental State Examination

MRI Magnetic resonance imaging

MSA Multisystem atrophy

mtDNA Mitochrondrial DNA

Parkinsonism Typical and atypical PD including Parkinsonian syndromes

PBMC Peripheral blood mononuclear cell

PD Parkinson's disease Abbreviation Dcilnition

PDS Parkinsonian syndrome (also Parkinson's disease syndrome)

PM Premutation expansion with the 5'UTR of the FMRl gene (56-200

CGG repeats)

UPDRS Unified Parkinson's Disease Rating Scale

BRIEF DESCRIPTION OF THE FIGURES

[0023] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0024] Figure 1A a graphical representation of differential FMRl mRNA expression in PBMCs of 3 patient groups manifesting Parkinsonism (PD) and healthy controls. In PD patients with GZ treated with L-dopa, FMRl mRNA levels were increased compared to: (i) healthy controls with normal CGG allele in FMRl **-p<0.001 ; (ii) PD patients carrying normal CGG size that were L-dopa treated - ## pO.001 ; (iii) PD patients with normal

CGG size that were L-dopa untreated^■■ - p<0.001. Note: each · represents an outlier.

[0025] Figure IB is a graphical representation of differential FMRl mRNA expression in EBV transformed lymphoblasts of three patient groups manifesting Parkinsonism (PD) and healthy controls. In PD patients with GZ treated with L-dopa, FMRl mRNA levels were decreased compared to (i) healthy controls with normal CGG size - **-p<0.001 ; (ii) PD patients carrying normal CGG size allele in FMRl that were L-dopa treated - ## p<0.001 In PD patients with normal CGG size allele in FMRl untreated with L-dopa, FMRl mRNA levels were decreased compared to (i) healthy controls with normal CGG size - **- pO.001 ; (ii) PD patients carrying normal CGG size allele in FMRl that were L-dopa treated - ## pO.001. Note: each · represents an outlier.

[0026] Figure 1C is a graphical representation of differential FMRP expression in EBV transformed lymphoblasts of three patient groups manifesting Parkinsonism (PD) and healthy controls. In PD patients with normal CGG size allele in FMRl untreated with L-dopa, FMRP levels were decreased compared to (i) healthy controls with normal CGG size in FMRl- **-p<0.001 , *-p<0.05; (ii) PD patients carrying normal CGG size allele in FMRl that were L-dopa treated - ## p<0.001. [0027] Figure 2A is a graphical representation of the relationship between the CGG expansion size and FMR1 mRNA expression in PBMCs with clinical phenotype measurements (I: UPDRS; II and IV: MMSE, III: Ataxia/ICARS) in a patient subgroup (n=10) with Parkinsonism carrying GZ and age matched controls patients with a normal CGG size allele in FMR1 (n=10).

[0028] Figure 2B is a graphical representation of the relationship between the CGG expansion size and FMR1 expression in PBMCs with clinical phenotype measurements in a patient subgroup with Parkinsonism carrying GZ (n=10) and age matched healthy controls carrying normal size alleles in FMR1 (n=15) and carriers of PM alleles in FMR1 with undefined phenotype (n=5). In PD patients with GZ, FMR1 mRNA levels were increased compared to healthy controls with normal CGG size - *-p<0.05.

[0029] Figure 3 is a graphical representation of differential CYC1 mRNA expression in (A) PBMCs and (B) EBV transformed lymphoblasts of three patient groups manifesting Parkinsonism (PD) and healthy controls. In healthy controls with normal CGG size allele in FMR1, CYC1 mRNA levels in PBMCs were significantly lower compared to (i) PD patients with GZ or PM treated with L-dopa, **-p<0.001 , (ii) PD patients carrying normal CGG size allele in FMR1 that were L-dopa treated - **- p<0.001 (iii) PD patients with normal CGG size allele in FMR1 that were L-dopa untreated- **- pO.001.

[0030] Figure 4A is a graphical representation of the relationship between the CYC1 mRNA expression in PBMCs with clinical severity measurement Unified Parkinson's Disease Rating Scale (UPDRS) in a patient subgroup with Parkinsonism carrying GZ (n=8) and age matched control patients that did not carry an expansion (n=8).

[0031] Figure 4B is a graphical representation of the relationship between the CGG expansion size and CYC1 mRNA expression in PBMCs with clinical phenotype measurements in a patient subgroup with Parkinsonism carrying GZ alleles (n=10) and age matched healthy controls carrying normal size alleles (n=15) and carriers of PM alleles with undefined phenotype (n=5). [0032] In PM patients with uncharacterized phenotype in relation to PD, CYC1 mRNA levels were increased compared to (i) healthy controls with normal CGG size in FMR1 allele **- pO.001 (ii) PD patients carrying GZ expansion in FMR1 allele # p<0.05 In PD patients with GZ expansion, CYC1 mRNA levels were increased compared to healthy controls with normal CGG size in FMR1 **- p<0.001.

[0033] Figure 5 is a graphical representation of differential Caspase 8 expression in (A) PBMCs and (B) EBV transformed lymphoblasts of three patient groups manifesting Parkinsonism (PD) and healthy controls.

[0034] In healthy controls patients with normal CGG size allele in FMR1 Caspase 8 mRNA levels were lower in EBV transformed lymphoblasts compared to (i) PD patients with GZ or PM treated with L-dopa, **-p<0.001, (ii) PD patients carrying normal CGG size allele in FMR1 that were L-dopa treated - *- p<0.05 (iii) PD patients with normal CGG size allele in FMR1 that were L-dopa untreated- *- p<0.05.

[0035] Figure 6 is a graphical representation showing the association of FMR1 mRNA with CGG expansion size and expression of DNMT1 and CYC 1 in the whole blood of 26 GZ carriers and four normal controls. Statistical analysis was conducted using the R statistical computing package version 1.9. FMR1, DNMT1 and CYC1 mRNA levels were examined using reverse real-time PCR. An aliquot of total RNA was isolated from 3 ml of peripheral blood using Tempus Blood RNA tubes (Loesch et al, J Med Genet 44:200-204, 2007). All samples were diluted to 6 ng/μΐ and reverse transcription was performed using the Multiscribe Reverse Transcription System, 50 U/μΙ as per manufacturer's instructions. The 7900HT Fast Real-Time PCR System (Applied Biosystems) was then utilized to quantify FMR1 , DNMT1 and CYC1, where all target genes were standardized GUS using the relative standard curve method as previously described. Samples were quantified in arbitrary units [au] in relation to a standard curve. [0036] Figure 7 is a graphical representation of the relationships between the CGG expansion size and Parkinsonism in a sample of the normal size and GZ allele male carriers identified through screening Australian males affected with PDS. (A) Boxplot representation of the differences in UPDRS between PDS non-carriers (CGG<40) and PDS carriers (CGG>40). (B) Robust regression of UPDRS versus CGG repeat size in the combined PDS sample.

[0037] Figure 8 is a graphical representation of expression levels of sense and antisense FMRl transcripts, and selected markers of mitochondrial dysfunction in peripheral blood of PDS small CGG expansion carriers (GZ/PDS), compared with disease controls (NS/PDS), and healthy controls (HC). (A) FMRl 5' and 3' mRNA levels in PBMCs; (B) ASFMRl mRNA levels in PBMCs; (C) CYC1 mRNA levels in PBMCs; (D) mtDNA ND1 levels in whole blood. PM/FXTAS is an affected carrier (85 CGGs); PMAJN is a carrier of unknown phenotype (170 CGGs). Both these carriers were ascertained through fragile X families. ** - p<0.05 compared to HC; ## - - p<0.05 compared to NS/PDS.

[0038] Figure 9 is a graphical representation of examples of relationships between the mitochondrial molecular measures in blood and neurological and neuropsychological measures in a sample of the normal size and GZ allele male carriers from Australian males affected with PDS. Plots from robust regression of (A) UPDRS versus CYC1 mRNA; (B) MMSE (measure of dementia and cognitive status) versus ND1 mtDNA quantity in the combined sample; (C) CGG size versus ND1 mtDNA quantity in GZ carriers only; (D) MMSE versus ND1 mtDNA quantity in GZ carriers and the one PM carrier. [0039] Figure 10 is a graphical representation of differential expression of ASFMRl in PBMCs of three patient groups manifesting Parkinsonism (PD) and healthy controls. In patients with GZ CGG size allele in FMRl , and treated with L-dopa, the ASFMRl mRNA levels were (i) significantly higher than in PD patients without GZ or PM also treated with L-dopa, *-p<0.05, (ii) significantly higher than in healthy controls. #-p<0.05. [0040] Figure 11 is a graphical representation of differential levels of NDl mitochondrial DNA in whole blood of three patient groups manifesting Parkinsonism (PD) and healthy controls. In patients with GZ CGG size allele in FMRl , and treated with L-dopa the mtDNA levels were (i) significantly lower than in PD patients without GZ allele treated with L-dopa, # -p<0.05, (ii) significantly lower than in healthy controls. *-p<0.05. In patients with normal size allele in FMRl, and untreated with L-dopa the mtDNA levels were (iii) significantly lower than in PD patients without GZ allele treated with L-dopa, # - p<0.05, (iv) significantly lower than in healthy controls. *-p<0.05.

DETAILED DESCRIPTION

[0041] The present invention provides a biomarker of a neurological disease condition, its level of clinical severity and its progression prior to or following therapeutic and/or behavioral intervention. The biomarker or its level of expression is detected in cells of a subject such as whole blood cells or its constituent cells.

[0042] Reference to whole blood and its constituents includes peripheral blood mononuclear cells (PBMCs).

[0043] Reference to Levodopa (L-dopa; L-3,4-dihydroxyphenylalanine; as a method of treatment also includes analogues of L-dopa).

[0044] Reference to a " neurological disease or condition" includes a disease or condition having symptoms or preliminary indicators of a condition within the disease spectrum of Parkinsonism. The term "Parkinsonism" includes typical and atypical Parkinson's disease (PD) as well as Parkinsonian syndromes including Parkinson's disease spectrum (PDS). The term "disease" includes a condition. A neurological condition may exist which is not clinically classified as a disease. Such conditions include behavioral characteristics, memory and cognitive levels. Hence, the neurological disease or condition of the present invention includes a neurodegenerative disorder as well as a disease or condition having symptoms of a pathology classified within the Parkinsonism spectrum, and other disorders involving dysregulation of the dopaminergic system, including but not restricted to dementia, schizophrenia, bipolar disorder, chronic depression and autism spectrum disorders. A "neurological condition" includes a neuropathological condition. The terms "condition" and "disease" may be used interchangeably throughout the subject specification.

[0045] In one embodiment, the neurological disease or condition is Parkinsonism and in another embodiment, the disease or condition is Parkinson's disease (PD). Reference to PD includes from mild Parkinsonism to classical PD. It also includes typical and atypical PD.

[0046] In one aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of FMRl and/or ASFMRl in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is an elevation in expression of FMRl and/or ASFMRl in whole blood and its constituents or a decrease in expression of FMRl in EBV-transformed lymphocytes in the presence of a CGG expansion associated with a GZ mutation.

[0047] Another aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of FMRl in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is a decrease in expression of FMRl mRNA and protein in EBV-transformed lymphocytes in the absence of any CGG expansion.

[0048] Yet another aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of CYC l or Caspase 8 in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is an increase in expression of CYCl or Caspase 8 in the presence of a GZ expansion. [0049] Still yet another aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of CYCl in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is a decrease in expression of CYC l in the absence of a GZ expansion.

[0050] Even still yet another aspect of the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of mitochrondrial complex III in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is an increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion.

[0051] Yet another aspect of the present invention provides an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of FMR1 sense and ASFMR1 antisense in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is an increase in expression of FMR1 sense and ASFMR1 antisense.

[0052] Still another aspect of the present invention relates to an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of mitochondrial dysfunction in cells from the subject or a diagnostic equivalent thereof wherein clinical symptoms are considered severe when there is an increase in mitochondrial dysfunction.

[0053] Yet another aspect of the present invention relates to an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of dysfunction of the dopaminergic system wherein clinical symptoms are considered severe when there is an increase in dopaminergic dysfunction.

[0054] Reference to FMR1 , ASFMR1 , CYC 1 and Caspase 8 includes genes or genetic regions as well as proteins or gene products.

[0055] An "increase in mitochondrial dysfunction" includes the measurement of markers of mitochondrial function or dysfunction; an example of which in the present embodiment is CYC1 expression.

[0056] Hence, the present invention contemplates an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of a genetic region or protein selected from FMR1 , ASFMRl, CYC l and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein clinical symptoms are considered severe when:

(i) there is an elevation in expression of FMR1 and/or ASFMRl in whole blood and its constituents or a decrease in expression of FMR1 mRNA and protein in EBV- transformed lymphocytes in the presence of a CGG expansion associated with a GZ mutation;

(ii) there is a decrease in expression of FMR1 and/or FMR1 protein (FMRP) in EBV- transformed lymphocytes in the absence of any CGG expansion; (iii) there is an increase in expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) there is a decrease in expression of CYCl in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is an increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls; (vi) there is an increase in expression of mitochrondrial complex III in EBV- transformed lymphocytes in the absence of a GZ expansion;

(vii) there is increased expression of FMR1 sense and/or ASFMR antisense transcripts; and/or

(viii) there is increased mitochondrial dysfunction and/or dopaminergic dysfunction. [0057] It is proposed herein that the decrease in FMR1 expression in the absence of CGG expansion is due to the dysfunction of the dopaminergic system, that may be reversed by supplementation of L-dopa or its analogs.

[0058] The present invention further provides an assay to assess the severity of symptoms associated with a neurological condition in a subject, the method comprising determining the level of expression of a gene selected from FMR1 , ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein clinical symptoms are considered less severe when:

(i) there is a low to no elevation in expression of FMR1 and/or ASF R1 in whole blood and its constituents or a low to no decrease in expression of FMR1 in EBV- transformed lymphocytes in the presence of a CGG expansion associated with GZ mutation;

(ii) there is a low to no decrease by expression of FMR1 expression in EBV transformed lymphocytes in the absence of any CGG expansion;

(iii) there is a low to no increase in expression of CYC1 in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion; (iv) there is a low to no decrease in expression of CYC1 in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is a low to no increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion;

(vi) there is normal expression levels of FMR1 sense and/or ASFMR1 antisense transcripts; and/or

(vii) there is a normal level of mitochondrial function and/or dopaminergic system function.

[0059] The "level of mitochondrial function" is conveniently determined by measuring mitochondrial DNA (mtDNA) levels of expression as well as markers such as CYC l or caspase. Other biomarkers may also be measured. [0060] Reference to treated patients or subjects includes and encompasses the treatment with L-3,4-dihydroxyphenylalanine [Levodopa] (L-dopa) and/or its analogs and/or derivatives and/or functional equivalents.

[0061] By "low to no increase or decrease" and the like is meant relative to a normal control or where appropriate a severely affected subject and/or a subject having treatment or no treatment. Reference to "whole blood and its constituents" includes individual blood cells such as PBMCs. Reference to "expression of mitochondrial complex III" includes expression of genes encoding or associated with mitochondrial complex III or the production of components of mitochondrial complex III.

[0062] CYCl (cytochrome CI) is a nuclear oxidative-phosphorylation gene expressed during periods of mitochondrial expansion (Li et al, 1996 supra).

[0063] The assay of the present invention is useful for monitoring treatment of a subject with a neurological condition.

[0064] Accordingly, the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of FMR1 and/or ASFMR1 or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is a decrease in expression of FMR1 mRNA and/or protein in whole blood and its constituents or an increase in expression of FMR1 and/or ASFMR1 in EBV-transformed lymphocytes in the presence of a GZ mutation. [0065] In another embodiment, the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of FMR1 or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is an increase in expression of FMR1 expression in EBV-transformed lymphocytes in the absence of any CGG expansion.

[0066] In yet another embodiment, the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of CYC1 and/or Caspase 8 or other markers of mitochondrial dysfunction or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is a decrease in expression of CYC1 in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion.

[0067] Still yet another embodiment, the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising a therapeutic protocol provided to the subject and then determining the level of expression of CYC1 or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is an increase in expression of CYC 1 in the absence of a GZ expansion.

[0068] Even yet another embodiment, the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of mitochrondrial complex III or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is a decrease in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion. [0069] Still another aspect of the present invention further contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising a therapeutic protocol provided to the subject with and then determining the level of expression of FMR1 sense and ASFMR1 antisense transcripts or a diagnostic equivalent thereof in cells of the subject wherein the treatment protocol is considered effective when there is a decrease in expression of FMR1 sense or ASFMR1 antisense relative to the levels prior to treatment or to levels in healthy controls.

[0070] Hence, the present invention contemplates a method for monitoring treatment of a neurological condition in a subject, the method comprising providing the subject with a therapeutic protocol and then determining the level of expression of a genetic region or protein selected from FMR1 , ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein the treatment protocol is considered effective when: (i) there is a decrease in expression of FMR1 and FMRP in whole blood and its constituents or an increase in expression of FMR1 in EBV-transformed lymphocytes in the presence of a GZ mutation;

(ii) there is an increase in expression of FMR1 expression in EBV-transformed lymphocytes in the absence of any CGG expansion;

(iii) there is a decrease in expression of CYC1 in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion; (iv) there is an increase in expression of CYC1 in EBV-transformed lymphocytes in the absence of a GZ expansion; (v) there is a decrease in expression of mitochondrial complex III in whole blood and its constituents in the absence of GZ expression; (vi) there is an increase in expression of mitochondrial complex III in EBV-transformed lymphocytes in the absence of a GZ expansion;

(vii) there is a decrease in or normalization of expression of FMR1 (sense), FMRP, ASFMR (antisense) transcripts and mtDNA levels to normal control levels; and/or

(viii) there are indicators of normalization of mitochondrial function and/or dopaminergic system function.

[0071] Similarly, the assay of the present invention may be employed to determine when a treatment is not effective. In such a situation, a change of medicinal or therapeutic protocol may be required and/or some behavioral modification or counselling may be recommended.

[0072] The level of expression of a biomarker such as FMR1 , FMRP, ASFMR1 , CYC 1 , Caspase 8, mitochrondrial complex III or mtDNA or a diagnostic equivalent thereof may be at the level of mRNA or protein or may be measured more indirectly such as by monitoring RNA degradation products or consequential epigenetic events. The assay in certain embodiments requires a determination of the length of CGG expansion (if any) and/or methylation profile or particular genetic loci or genomic regions. The assay may also be cell type specific. For example, FMR1 or ASFMR1 gene expression may be increased in whole blood and its constituents but decreased in EBV-transformed lymphocytes. Another variable may relate to whether the subject is or has undergone therapeutic treatment. [0073] Although not intending on limiting the present invention to any one theory or mode of action, it is proposed that subjects having a PM or GZ mutations exhibit significant increase in FMR1 mRNA levels relative to a normal control which contributes to cytoxicity and elevates the severity of neurological involvement in subjects affected with a neurological disease or condition such as but not limited to Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and autism spectrum disorders. Furthermore, it is proposed that neuropsychological and neurological changes are more severe in carriers of such PM/GZ alleles relative to non-carriers. In addition, FMR1 and ASFMR1 expression reflects a subject's likely response to Levodopa (L-dopa) treatment in neurodegenerative disorders included within the neurological condition. Levels of mitochondrial function or extent of dysfunction determined, for example, by determining mtDNA expression levels including CYC1 expression levels, also determine the severity of a disease condition.

[0074] Similarly, it is proposed that subjects with PM or GZ mutations exhibit higher levels of expression of CYC1 and Caspase 8 and this contributes to clinical severity.

[0075] It is apparent, therefore, that the present invention provides a biomarker that correlates with the severity of symptoms of the neurological condition and/or its progression, wherein: (a) Patients with the neurological condition that have a small CGG repeat expansion in FMR1 gene defined as GZ (40-55 repeats) have abnormal FMR1 mRNA compared to age matched controls (healthy and Parkinsonism without an expansion in FMR1 ). The abnormal FMR1 mRNA expression whole blood or its constituents, is associated with potential neurodegeneration as reflected in worsening clinical symptoms, (e.g. an increase in Unified Parkinson's Disease Rating Scale (UPDRS)) and MRI changes-which are manifested by general brain atrophy and, more specifically, the white matter degeneration in certain brain areas which can be seen on the images or more precisely assessed using volumetric measures; moreover, Multinuclear Magnetic Resonance Spectroscopy can be used for in vivo Assessment of Mitochondrial Dysfunction (b) Patients with 40 to 55 CGG repeats expansions in FMRl respond poorly to L-dopa treatment

(c) The response to L-dopa treatment is inversely correlated with an increase in FMRl and ASFMRl mRNA in PBMC's and there is a decrease in FMRl mRNA and FMRP in

EBV transformed lymphoblast. There are also relative changes in the brain of these markers.

(d) Patients with a neurological condition without an expansion in FMRl untreated compared to age matched treated controls have a decrease in FMRl mRNA and FMRP in

EBV transformed lymphoblasts. FMRl mRNA and FMRP and epigenetic modification of FMRl promoter may indicate the level of response to treatment.

(e) Patients with a neurological condition that have an expansion in FMRl defined as GZ (40-55 CGG repeats) or premutation (56-200 CGG repeats) have an increase in

Cytochrome C I mRNA and iso-1 -cytochrome C protein expression in whole blood or its constituents (both are associated with the mitochondrial complex III) compared to age matched controls (either healthy, or neurologically challenged without an expansion in FMRl ). The increased CYC 1 expression is associated with the severity of neurological involvement, increase in UPDRS, and potentially with the level of white matter degeneration assessed by MRI, including volumetric measures. Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurological pathology. (f) Patients with a neurological condition that have an expansion in FMRl defined as GZ (40-55 CGG repeats) or premutation (56-200 CGG repeats) have an increase in mitochondrial complex III RNA and protein expression in whole blood or its constituents compared to age matched controls (healthy and neurologically challenged without an expansion in FMRl). The increased mitochondrial complex III RNA and protein expression is associated with potential neurodegeneration as reflected in severity of clinical symptoms, (e.g. increase in UPDRS) [ and potentially with the level of general brain atrophy, especially white matter degeneration assessed by MRI, including volumetric measures]. Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurological pathology.

(g) Patients with a neurological condition that do not carry expansion in FMR1 have increased Cytochrome CI mRNA and iso-1 -cytochrome C protein expression in whole blood or its constituents compared to age matched controls (healthy without an expansion in FMR1). The increased CYC1 expression is associated with severity of neurological involvement, (e.g. increase in UPDRS and potentially with the level of general brain atrophy, especially white matter degeneration assessed by MRI, including volumetric measures). Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurological pathology.

(h) Patients with a neurological condition that do not carry an expansion in FMR1 have increased mitochondrial complex III mRNA and protein expression in whole blood or its constituents compared to age matched controls (healthy without an expansion in FMR1 ). The increased activity of mitochondrial complex III mRNA and protein is associated with the severity of neurological involvement, (e.g. an increase in UPDRS, and potentially with the level of general brain atrophy, especially white matter degeneration assessed by MRI, including volumetric measures). Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurological pathology.

(i) Patients with a neurological condition have an increase in Caspase 8 mRNA and protein expression and activated forms in EBV transformed lymphoblasts compared to age matched controls (healthy). The increased Caspase 8 activity is associated with the severity of neurological involvement, (e.g. increase in UPDRS [and potentially with the level of general brain atrophy, especially white matter degeneration assessed by MRI, including volumetric measures]). Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurolological pathology.

(k) Patients that have a small expansion (40-55) in FMR1 gene and an increase in CYC 1 mRNA and iso-1 -cytochrome C protein in whole blood or its constituents is associated with the severity of neurological involvement, increase in UPDRS, (and potentially with the level of general brain atrophy, especially white matter degeneration assessed by MR1, including volumetric measures). Additionally, Multinuclear Magnetic Resonance Spectroscopy may provide in vivo Assessment of Mitochondrial Dysfunction, which is likely associated with neurological pathology.

[0076] Reference to "UPDRS" means Unified Parkinson's Disease Rating Scale. Reference to "whole blood and its constituents" includes PBMCs. [0077] The present invention extends to diagnostic equivalents of the biomarkers listed. For example, increased or decreased FMR1 and ASFMR1 expression may lead to consequential changes in expression of other members of the FMR genetic locus or in levels of expression of other genetic loci. Similarly, CYC1 expression levels may be substituted by expression levels of other genes in the election transport chain including NADPH oxidase and an increase in production of reactive oxidative species.

[0078] Standard assays may be employed to determine expression levels including measuring mRNA levels, promoter levels, protein levels and consequential expression levels of affected genetic loci.

[0079] For the determination of epigenetic patterns, any methylation assay may be employed such as methylation specific melting curve analysis (MS-MCA) or high resolution melting (MS-HRM) [Dahl et al, Clin Chem 55^:790-793, 2007; Wojdacz el al, Nucleic Acids Res. 35(6):e4\ , 2007]; quantification of CpG methylation by MALDI-TOF MS (Tost et al, Nucleic Acids Res 3J(9):e50, 2003); methylation specific MLPA (Nygren et al, Nucleic Acids Res. 33(14) e\2$, 2005); methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) [Yegnasubramanian et al, Nucleic Acids Res.

9, 2006] or methylation sensitive oligonucleotide microarray (Gitan et al, Genome Res. 72( ^: 158-164, 2002), as well as via antibodies. [0080] Insofar as the assays of the present invention involve an amplification, any amplification methodology may be employed. Amplification methodologies contemplated herein include the polymerase chain reaction (PCR) such as disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195; the ligase chain reaction (LCR) such as disclosed in European Patent Application No. EP-A-320 308 and gap filling LCR (GLCR) or variations thereof such as disclosed in International Patent Publication No. WO 90/01069, European Patent Application EP-A-439 182, British Patent No. GB 2,225, 1 12A and International Patent Publication No. WO 93/00447. Other amplification techniques include Q replicase such as described in the literature; Stand Displacement Amplification (SDA) such as described in European Patent Application Nos. EP-A-497 272 and EP-A-500 224; Self-Sustained Sequence Replication (3SR) such as described in Fahy et al, PCR Methods Appl. ](J):25- 33, 1991 and Nucleic Acid Sequence-Based Amplification (NASBA) such as described in the literature.

[0081] A PCR amplification process is particularly useful in the practice of the present invention.

[0082] A "nucleic acid" as used herein, is a covalently linked sequence of nucleotides in which the 3' position of the phosphorylated pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide and in which the nucleotide residues are linked in specific sequence; i.e. a linear order of nucleotides. A "polynucleotide" as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length. An "oligonucleotide" as used herein, is a short polynucleotide or a portion of a polynucleotide. An oligonucleotide typically contains a sequence of about two to about one hundred bases. The word "oligo" is sometimes used in place of the word "oligonucleotide". The term "oligo" also includes a particularly useful primer length in the practice of the present invention of up to about 10 nucleotides. [0083] As used herein, the term "primer" refers to an oligonucleotide or polynucleotide that is capable of hybridizing to another nucleic acid of interest under particular stringency conditions. A primer may occur naturally as in a purified restriction digest or be produced synthetically, by recombinant means or by PCR amplification. The terms "probe" and "primers" may be used interchangeably, although to the extent that an oligonucleotide is used in a PCR or other amplification reaction, the term is generally "primer". The ability to hybridize is dependent in part on the degree of complementarity between the nucleotide sequence of the primer and complementary sequence on the target DNA.

[0084] The terms "complementary" or "complementarity" are used in reference to nucleic acids (i.e. a sequence of nucleotides) related by the well-known base-pairing rules that A pairs with T or U and C pairs with G. For example, the sequence 5 -A-G-T-3' is complementary to the sequence 3'-T-C-A-5' in DNA and 3 -U-C-A-5' in RNA. Complementarity can be "partial" in which only some of the nucleotide bases are matched according to the base pairing rules. On the other hand, there may be "complete" or "total" complementarity between the nucleic acid strands when all of the bases are matched according to base-pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands as known well in the art. This is of particular importance in detection methods that depend upon binding between nucleic acids, such as those of the invention. The term "substantially complementary" is used to describe any primer that can hybridize to either or both strands of the target nucleic acid sequence under conditions of low stringency as described below or, preferably, in polymerase reaction buffer heated to 95°C and then cooled to room temperature. As used herein, when the primer is referred to as partially or totally complementary to the target nucleic acid, that refers to the 3 '-terminal region of the probe (i.e. within about 10 nucleotides of the 3'-terminal nucleotide position).

[0085] Reference herein to a stringency in relation to hybridization includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C) % (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C. [0086] As used herein, the terms "subject", "patient", "individual", "target" and the like refer to any organism or cell of the organism on which an assay of the present invention is performed whether for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include both male and female humans but the present invention extends to experimental animals such as non-human primates, (e.g. mammals, mice, rats, rabbits, pigs and guinea pigs/hamsters). The "subject" may also be referred to as a population since the present invention is useful in population studies including epidemiological studies or assays of an ethnic population.

[0087] The present invention also contemplates kits for determining the level of expression of one or more genetic loci within the genome of a eukaryotic cell or group of cells. The kits may comprise many different forms but in one embodiment, the kits comprise reagents for the determination of gene expression at the mRNA or protein level.

[0088] A further embodiment of the present invention is a kit for the use in the above methods comprising primers to amplify a site within or proximal to a target biomarker. The kit, for example, can be used to determine clinical severity of a subject with a neurological condition wherein:

(i) there is an elevation in expression of FMRl and/or ASFMRl in whole blood and its constituents or a decrease in expression of FMRl in EBV-transformed lymphocytes, in the presence of a CGG expansion associated with a GZ mutation;

(ii) there is a decrease in expression of FMRl in EBV-transformed lymphocytes in the absence of any CGG expansion; (iii) there is an increase in expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) there is a decrease in expression of CYCl in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is an increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion;

(vi) an increase in FMRl sense and/or ASFMRl antisense expression corresponds to a more severe clinical situation; and/or

(vii) mitochondrial dysfunction and/or dopaminergic dysfunction correspond to degree of clinical severity of disease.

[0089] The kit may also comprise instructions for use. [0090] Conveniently, the kits are adapted to contain compartments for two or more of the above-listed components. Furthermore, buffers, nucleotides and/or enzymes may be combined into a single compartment. [0091] As stated above, instructions optionally present in such kits instruct the user on how to use the components of the kit to perform the various methods of the present invention. It is contemplated that these instructions include a description of the detection methods of the subject invention, including detection by gel electrophoresis, real-time PCR and Western blot analysis.

[0092] The present invention further contemplates kits which contain a primer for a nucleic acid target of interest with the primer being complementary to a predetermined nucleic acid target. In another embodiment, the kit contains multiple primers or probes, each of which contains a different base at an interrogation position or which is designed to interrogate different target DNA sequences. In a contemplated embodiment, multiple probes are provided for a set of nucleic acid target sequences that give rise to analytical results which are distinguishable for the various probes. The multiple probes may be in microarray format for ease of use. [0093] The present invention is now described by the following non-limiting Examples.

EXAMPLE 1

Data sources and procedures

[0094] All subjects were initially screened for the size of the FMRl CGG repeat using PCR amplification of DNA extracted from saliva samples (Table 2). In addition to non- carriers with Parkinson's Disease Spectrum (PDS) including typical/atypical PD or Parkinsonian symptoms such as MSA, 18 healthy controls matched for age and sex were recruited from amongst patients' relatives and friends to provide the baseline molecular data. The existence of any serious medical condition or neurological problems in the control group was ruled out by detailed medical history and general neurological examination, and saliva samples collected and DNA analyzed for CGG repeat size to confirm the non-carriers' status. Once this was confirmed, blood samples were collected from patients and controls for the molecular testing. Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll separation and EBV-transformed lymphoblasts were generated. These samples were examined for: (i) the levels of FMRl and ASFMRl mRNA and its protein product (FMRP); (ii) CYC 1 and Caspase 8 mRNA levels; (iii) mitochrondrial complex III (iv) ND1 mitochondrial DNA quantity. All patients affected with Parkinsonism carrying GZ expansions greater than 41 CGG repeats (n=10) and age matched controls patients with Parkinsonism but without an expansion in the FMRl (n=10) were subjected to further clinical testing, which included comprehensive neurological, neuropsychological and magnetic resonance imaging (MRI) [including volumetric measures] assessments.

[0095] The effect of the small expansion alleles on clinical and molecular parameters was deduced from the comparison of the results from the GZ/PM carriers with the same data from the group of patients affected with Parkinsonism with normal CGG repeat sizes treated and untreated with L-dopa, and from healthy control non-carriers matched for age and sex. [0096] The effect of treatment on clinical and molecular parameters was obtained from the comparison of the results from the group of patients affected with Parkinsonism with normal CGG repeat sizes treated with L-dopa, with the same data from the group of patients affected with Parkinsonism with normal CGG repeat sizes untreated with Levodopa (L-dopa) , and from healthy control non-carriers matched for age and sex.

Table 2

EXAMPLE 2

Protocols

[0097] Sample Processing. Initial CGG repeat size screening was based on DNA extracted from saliva samples (Oragene-DNA tubes, DNA Genotek Inc.). PBMCs were isolated from blood using Ficoll gradient separation (Amersham Pharmacia Biotech). PBMCs were then split into three equal fractions for: (i) DNA extraction using a BIO ROBOT M48 DNA Extractor (Qiagen Inc., Hilden, Germany) for CGG repeat size confirmation and FMR1 promoter methylation analyzes; (ii) RNA extraction (Rneasy kit; Qiagen) for gene expression analyses; (iii) standard EBY transformation. [0098] CGG repeat size using PCR amplification. This was assessed by a fully validated PCR assay using primers c and f (Maddalena et al, Genet Med i:200-205, 2001), with a precision of +/- one triplet repeat across the normal and GZ ranges achieved by using a fragment analyzer (MegaBace, GE Healthcare), with appropriate normal and pathological (expanded) controls.

[0099] Transcript levels for FMR1-573', ASFMR1 , CYC1 representing a component of mitochrondrial complex III and Caspase 8 using real time PCR. Reverse transcription using the Multiscribe Reverse Transcription System (Applied Biosystems) was performed on samples of high total RNA quality, as assessed by the Experion capillary electrophoresis system as previously described (Godler et al, BMC Clin Pathol 9:5, 2009).

[0100] Total RNA was extracted, purified and reverse transcribed. The 7900HT Fast Real Time PCR (Applied Biosystems) was used to quantify FMRl -5', FMR1-3', ASFMRl , GAPDH, B2M, and GUS, using the relative standard curve method. DNase treatment of total RNA was found to significantly affect levels of ASFMRl as well as FMRl and thus was not used for these assays in the study. The target gene and the internal control gene dynamic linear ranges were performed on a series of doubling dilutions of an RNA standard (160-4 ng/ul). A no reverse transcription enzyme control was included for every sample for ASFMRl (since this assays did not target an exon/exon boundary). The difference between the plus and minus no reverse transcriptase control was considered as the ASFMRl expression value for each sample. Previously published sequences were used for primers and probe for: FMR1 -5' and GUS (Tassone et al, Am J Hum Genet. 66(1):6- 15, 2000); FMRl-3'(Pietrobono et al, Hum Mol Genet. 14(2):2βΊ-2ΊΊ , 2005); ASFMRl (FMR4) [Khalil et al, PLoS ONE. 3(%⁾:el486, 2008]. FMR1-5', FMR1-3 ', ASFMRl ' primers and probes were be used at concentrations of 18μΜ and 2μΜ, respectively. EIF4A2 and SDHA primer/probe mixes were be obtained from PrimerDesign (PerfectProbe ge-PP-12-hu kit) and used at concentration of 2μΜ. All of the above assays were single-plexed, with each sample assayed in duplicate 10 μΐ PCR reactions. The reactions consisted of 5.8 mM MgCl₂, 1 μΐ Buffer A (Applied Biosystems), 3.35 μΐ RNase- free water, 1.2 mM dNTPs, 0.01 units/μΐ of AmpliTaq Gold, 0.5μ1 of TaqMan probe and 0.5μ1 forward and 0.5μ1 reverse primers, and Ι μΐ of the reverse transcription (cDNA) reaction. The annealing temperature for thermal cycling protocol was 60°C for 40 cycles. The samples were quantified in arbitrary units (au) in relation to the standard curves performed on each plate, standardized to the mean of the three internal control genes (GUS, EIF4A2 and SDHA).

[0101] Standard Western blot analysis. This protocol was utilized for FMRP quantification performed on 20 μg of total protein lysate. Standard curves were included on each blot. The goat anti-mouse HRP conjugated antibody (Jackson Immuno Research) was as secondary antibody for FMRP analysis, using primary antibody 1 C3 from N-terminal FMRP common region (Chemicon). Actin was used as a loading control. An ECL system (GE Healthcare) was used to develop the blot, and the results expressed as mean band density ratios between total target protein and actin, determined using ImageQuant software (Molecular Dynamics).

[0102] Clinical assessments and neuropsychological testing. Apart from standard medical history and general neurological examination, tremor, ataxia, and Parkinsonism scales were administered and videotaped according to standard protocol (Berry- rvais et al, Ann Neurol 53:616-623, 2003), which incorporates the Clinical Rating Scale for Tremor, CRST (sections A,B,C), International Cooperative Ataxia Rating Scale, ICARS, and the Unified Parkinson's Disease Rating Scale (UPDRS) [Version 2.0 1991-Motor examination III, items 18-44]. [0103] Standard cognitive functions were assessed using the Wechsler intelligence test (WAIS-III), appropriate for individuals over the age of 16 (Wechsler (ed), The Wechsler Adult Intelligence Scale-Third Edition: Adminsitration and Scoring Manual, (Orlando: The Psychological Corporation), 1997). Higher cognitive (executive) functioning were assessed using: the Wisconsin Card Sorting test (WCST), which measures cognitive set-shifting ability (Heaton, Wisconsin Card Sorting Test Manual: Revised and expanded [TX.Psychological Assessment Resources, Odessa], 1993); the Stroop Color and Word Test (STROOP) which measures selective attention, cognitive flexibility, and the ability to inhibit automatic responses (Trenerry, Stroop Neuropsychological Screening Test [Psychological Assessment Resources, Florida], 1988); and the Symbol Digit Modalities Test (SDMT) which assesses participants' processing speed and tracking ability, independently of motor control impairments (Smith, Learn Disord 3:83-91 , 1968).

[0104] Addenbrooke's Cognitive Examination Final Revised Version A, ACE-R (Mathuranath et al, Neurology 55(77^: 1613-1620, 2000), was also used to screen for cognitive impairment. The total score (ACE-R Full) was a raw score derived from the sum of the five sub-scores, measuring attention/orientation, memory, fluency, language and visuospatial cognitive domains. A full Mini-Mental State Examination (MMSE) is embedded within the ACE-R, and is particularly useful in the assessment of patients with dementia (Kukull et al, J. Clin. Epidemiol. 54(5^: 1050- 1058, 1994). Processing speed was measured using the Symbol Digit Modalities Test (SDMT) [Smith, Symbol Digit Modalities Test, Los Angeles, CA, USA, Western Psychological Services, 1991 ]. The testings were conducted by a trained neuropsychologist who was not aware of the participants' carrier status at the time of assessment.

[0105] High resolution MR imaging and volumetric measures. Both descriptive and volumetric analysis of the MR images was carried out at neuroimaging laboratories using a uniform protocol. MRI were obtained using a 3-D coronal gradient echo volume acquisition sequence as well as routine axial and coronal T2 and proton density weighted images. Axial and coronal TSE T2 and PD, axial and coronal MPRS, and coronal Tl MPR volume whole brain imaging were acquired on a Siemens Avanto 1.5T MRI (St. Vincent's), or a GE 1.5T MRI (Alfred). It has been previously shown that adequate and comparable images are obtained on both machines. A Tl weighted MP-Rage volumetric sequence is obtained (matrix dimensions 256 X 192, Field Of View 200 X 150 mm, slice thickness 1.5 mm) in addition to routine sequences. Volumetric analysis are performed on a UNIX workstation using ANALYZE (Trademark) AVW (BIR, Mayo Clinic, Rochester USA). The contiguous Tl weighted volumetric sequence scans are used in the extraction of whole brain volumes based upon a series of 3D morphological operations, as described in the ANALYZE (Trademark) AVW User's Guide. Details relating to the measurement techniques of specific intracranial structures were previously outlined and validated (Cook, Acta Neurol Scand 752: 109-1 14, 1994; Lawson et al, 2000 supra). The results were compared with the data from controls from an MRI databank. EXAMPLE 3

Statistical analysis

[0106] A t-test or a rank test was used to assess the differences in the levels of FMR1 and ASFMR1 transcripts, FMRP and mitochondrial and pro-apoptotic markers CYC 1 and Caspase 8 and neurological and neuropsychological scores between the carrier, and two non-carrier groups. The FMR1 and ASFMR1 mRNA levels (explanatory variables) were also related to the rate of apoptosis (response variables: CYC1 and Caspase 8 levels) and FMRP, using multiple linear regression, and adjusting for the confounders, such as age, sex, and treatment. The analysis was a straightforward significance test in a linear regression. The clinical impact of molecular pathology was be examined using multiple regression, where the response variables were be neurological and psychological scores, and the explanatory variables were the FMR1 and ASFMR1 mRNA, FMRP and apoptotic measures CYC 1 and Caspase 8. Canonical correlation was also used to determine which combination of the molecular/apoptotic variables best explained the combinations of clinical responses.

[0107] The data were also analyzed using STATA statistical software (version 10.0, StataCorp, College Station, TX, USA) and plotted using the publically available R package {A language and environment for statistical computing [computer program]. Version: R Foundation for statistical computing; 2009). Because of the sample size and the non- Gaussian distributions, the measures were compared from the sample of carriers with those from non-carriers using the nonparametric ruskall-Willis test. In order to down-weight the effect of outliers robust regression was used to model relationships between major phenotypic and molecular measures. Age adjustment was applied whenever appropriate, but illness duration was no a significant confounder.

[0108] The results of regression in GZ/PM carriers were of the main interest, but non- carriers were analyzed to obtain baseline data. Measurement error models were adopted (Lenth, Java Applets for Power and Sample Size [Computer software], http://www.stat.uiowa.edu/~rlenth/Power., 2006:2009) to adjust for extensive variability in any of the explanatory variables. The analyses were conducted using the publicly available R statistical computing package, version 1.191 (R development Core team, 2004). The results are shown in Tables 3 through 5.

Table 3

FMR1/FMRP

Table 4

CYCI/Caspase 8/mitochondrial DNA

Table 5

Subset of patients with idiopathic PD in PBMCs.

(Pairwise correlation matrix)

Key to tables:

Parkinsonism/UPDRS - Unified Parkinson's Disease Rating Scale, UPDRS Clinical tremor / CSRT - General scale to measure tremor

ATAXIA/ICARS - Federation of Neurology has proposed a one-hundred-point semiquantitative International Cooperative Ataxia Rating Scale (ICARS). The scale proposed involves a compartimentalized quantification of postural and stance disorders, limb ataxia, dysarthria and oculomotor disorders, in order that a subscore concerning these symptoms may be separately studied.

MMSE: Mild-Moderate-Severe: Mild dementia falls into the Mini Mental Status Exam (MMSE) 20-24 range, moderate 10-19 range, and severe below 10. Moderately demented patients typically need help with activities of daily living, while severely demented are totally dependent. A useful measuring tool for clinicians for dementia is the Mini Mental Status Exam. However, the MMSE does not differentiate depression in the elderly from mild cognitive impairment, although it successfully differentiated MCI patients from normal elderly or dementia (see, Primary Care Companion J Clin Psychiatry. 7(2):62-69, 2005). The MMSE correlates well with the cognitive scale of the Alzheimer's Disease Assessment Scale (ADAS), the standard cognitive measure in drug research studies.

ACER-Fw - IQ and dementia measure Dementia (yes or no)

HVTLE-Iearning - test for executive functioning skills.

EXAMPLE 4

Linking the FMRl alleles with small CGG expansions with neuro development disorders

[0109] A comprehensive molecular testing was conducted in 26 consenting Caucasian male carriers of GZ alleles aged 7-19 years from the total sample of 43 carriers identified by Mitchell et al, Clin Genet 67:38-46, 2005. The CGG repeat number ranged from 41 to 54, with one subject with 55 repeats also included as borderline. The majority (16) came from the SEN population, five carriers were from a non-SEN population from the same schools, 5 were brothers of the identified carriers. A lower boundary of 40 CGG repeats was adopted for the GZ range based on earlier finding that the onset of an elevation of mRNA levels was recorded in FMRl alleles with 40 repeats (Loesch et al, 2007 supra). Four normal repeat size controls were included in molecular analyses. The neuropsychological testing was conducted in all but two GZ carriers (N=24). Six participants had their FSIQ scores (assessed by the Wechsler Intelligence Scale appropriate for age) < 1 SD, but nearly a half (1 1/24) showed deficit in executive skills assessed by The Controlled Oral Word Association Test (Spreen & Benton, Neurosensory Center Comprehensive Examination for Aphasia, 1977), seven out of 20 showed impairment in adaptive skills assessed by the Vineland Adaptive Behavior Scales Interview (Sparrow & Cicchetti, Appl Res Ment Retard 5:219-231 , 1984), and a half showed concentration deficits assessed by The Conner 's Global Index-Parent version (Conners et al, Psychopharmacol Bull 52:67-73, 1996). One third of the sample was classified as ASD using the Autism Diagnostic Observation Schedule-Generic, ADOS-G (Lord et al, Autism Diagnostic Observation Schedule Los Angeles: Western Psychological Services, 1999). [0110] The major aim of this study was to get an insight into the mechanisms involved in the FMRl mRNA 'toxicity' in GZ by relating the levels of mRNA transcript to the expression of a prominent epigenetic marker, DNA-methyltransferase 1 (DNMT1). The results are shown in Figures 1 through 6. This marker is known to regulate FMRl transcription in full mutation subjects (Pietrobono et al, Nucleic Acids Res 30(14):3278- 3285, 2002) and to play a major role in regulation of global methylation coupled to DNA replication (Biniszkiewicz et al, Mol Cell Biol 22:2124-2135, 2002). The relationship was also investigated between levels of these two transcripts and Cytochrome CI (CYCl), a nuclear oxidative-phosphorylation gene expressed during periods of mitochondrial expansion (Li et al, 1996 supra). This was to explore the possibility that epigenetic mechanisms are associated in the FMRl mRNA toxicity of GZ alleles, leading to CYC l over expression associated with mitochondrial dysfunction (Li et al, 1996 supra). The methods of CGG repeat sizing and FMRl mRNA assessments were reported in: Loesch et al, 2007 supra. The assays for FMRl mRNA levels are validated using a relative standard curve method. [0111] The results of correlations between the molecular measures assessed using a linear regression showed significant relationship between the levels of FMRl mRNA and CGG size p=0.046 (Figure 6A), DNMTl p=0.022 (Figure 6B), and CYCl p=0.045 (Figure 6C). The DNMTl expression was also significantly correlated with CYC l expression (Figure 6D). The relationship between CGG size and DNMTl or CYC l levels was not significant, but an extensive variability and the small sample size should be considered. The correlations between neuropsychological scores and molecular measures, using regression models appropriate for the shape of distribution of individual variables, were not significant in the reduced sample of 24 (or less for some neuropsychological measures). [0112] Since ASD was one of the commonest diagnoses in the identified SEN carriers, an independent sample of ASD children was screened, diagnosed using ADOS-G (Lord et al, 1999 supra), for the size of CGG repeats in the FMRl gene. This sample comprised 42 Caucasian male children and adolescents aged 5-20 years, recruited via advertisements placed in publications of autism specific organizations in Victoria. Potential heterogeneity of the sample was reduced by selecting higher functioning individuals based on their performance on a Wechsler intelligence test, with the median PIQ of 89, and omitting individuals with co-morbid diagnoses. All participants provided cheek swab samples. Genomic DNA was obtained by placing a buccal brush in a labeled tube containing 400 μί of 50 mM NaOH, rotating, and incubating at 95°C for 15 minutes. 80 of 1 M Tris-HCl pH 7.5 was added, mixed and centrifuged, and then stored at 4°C. The supernatant was removed and stored at -20°C until testing. The CGG repeat sizing was performed using a fully validated PCR assay (Khaniani et al, 2008 supra), and was assessed with precision of +/- one triplet repeat across the normal and GZ ranges, using a fragment analyser (MegaBace, GE Healthcare). The distribution of the number CGG repeats from the autism sample was compared with a distribution in a population of Tasmanian newborns (described in Mitchell et al, 2005 supra), where DNA was extracted from autoclaved Guthrie cards following the procedure described by Holden et al, Am J Med Genet 64:3 \ 3- 318, 1996. Scoring of the CGG repeat size was as in: Holden et al, 1996 supra using the same primers c and / (Fu et al, Cell (57: 1047-1058, 1991 ) in a radioactive reaction. PCR products, together with allele ladders of known sizes, were run on a 6% w/v polyacrylamide gel. The results (triplet repeat number) of this test correlated very highly with the results obtained from the saliva samples (r²=0.974, y=0.999x-0.108), which shows the robustness of the analyses. A significant excess of GZ carriers ( =5, with repeat size ranging from 41-52) was found in the autism sample of 42, compared with a normal control sample of 576, with 19 carriers identified (two sided Fisher's exact test p-value =0.018).

[0113] The results indicate that GZ alleles are associated with some behavioral phenotypes, including ASD, and that epigenetic dysregulation, triggered by elevated FMR1 transcript levels is involved in the origin of these phenotypes. Moreover, a significant elevation of the FMR1 transcript was shown and there was a linear relationship between the levels of this transcript and the number of CGG repeats in alleles within the GZ range. The findings here are consistent with the earlier data based on human cell lines transfected with the FMR1 5'-UTR containing CGG repeat lengths ranging 0 to 99, and a downstream reporter gene, which demonstrated an increase in transcription levels for constructs possessing either PM or GZ alleles (Chen et al, Hum Mol Genet 72:3067-3074, 2003).

[0114] Further, it was found that the over-expression of FMR1 was significantly correlated with the expression of DNMTl , and there was a marginally significant relationship between the expression levels of DNMTl and CYC1 , the latter being a marker of mitochondrial dysfunction (Li et al, 1996 supra; Ayub & Hallett, Aging Cell 5 (¾): 145-149, 2004). These findings are suggestive of epigenetic involvement in GZ alleles, and are supportive of the results obtained for PM alleles from a human kidney fibroblast cell line (HEK293), where insertion of 176 CGG repeats 3' of the FMRl promoter induced over- expression of FMRl mRNA, and altered expression of major epigenetic factors, which was associated with increased apoptosis (Handa et al, FEBS Lett 579, 2702-2708, 20050].

[0115] The cognitive and behavioral status of 24 GZ carriers included in this study were characterized and found that the most common manifestations were impaired executive skills and attention problems, and one third fell into the ASD category. These findings are suggestive of the role of GZ alleles in the origin of these phenotypes.

[0116] In summary, the data indicate that an epigenetic component is involved in a "toxicity" of excessive FMRl and/or ASFMR1 transcript levels in small expansion alleles, leading to decreased cell survival and thus contributing to the increased risk of neurodevelopmental conditions, including autism. This indicates a role for epigenetic therapy.

EXAMPLE 5

Toxicity of bidirectional transcripts and mitochondrial dysfunction with small CGG expansions in FMRl gene [0117] The hypothesis that CGG expansion and possibly the associated molecular changes are significantly related to the severity of Parkinsonian manifestations, including the rate of cognitive decline was tested. Strong evidence for the effect of CGG repeat on the these manifestations, based on data from 14 GZ/PM carriers and 24 non-carriers, was provided by the finding that UPDRS motor score was significantly elevated in the sample of GZ/PM carriers compared with PDS non-carriers (Figure 7A), and that this score was also significantly related to CGG repeat size (Figure 7B). The CGG expansion size was also significantly correlated with MMSE score representing cognitive decline, and reaction time (RT) assessed by CATSYS (Table 6). That the severity of neurological involvement is also affected by other relevant changes in small expansion FMRl alleles has been shown by the results of regression analyses which included the levels of both sense and antisense FMRl transcripts and explanatory measures, and several Parkinsonian motor measures and cognitive scores, as dependent variables (Table 6A). Despite small sample size, the levels of both these transcripts were significantly elevated in the PM/GZ carriers with PDS compared with non-carriers (Figures 8A and B), and showed marginally significant associations with UPDRS. Notably, although FMRl was not significantly associated with other clinical measures, ASFMR1 showed significant correlation with all but one measure (CATSYS-RT).

[0118] Since the PDS carrier and non-carrier samples were group-matched for disease duration and age, it was not unexpected to find no relationship between the duration and any of the clinical and neuropsychological measures. However, both measures of dementia (MMSE and ACER) were found to be age-related and were thus adjusted for age in all the regressions analyses involving those two variables. [0119] CYC1 expression analysis was included in order to determine a set of the most stable reference genes for an assessment of the FMRl mRNA levels from a set described in Vandesompete et al, Genome Biol 3( ):RESEARCH 0034, 2002, using geNorm algorithm. However, unlike any other reference genes included in the relevant kit (PrimerDesign kit PerfectProbe ge-PP-12-hu kit), the CYC1 gene showed differences in expression between the carrier and control groups (Figure 8C). More specifically, the CYC1 transcript was found to be elevated in GZ/PM carriers (GZ/PDS) relative to healthy controls (HC), and the levels of this transcript showed a highly significant relationship with the severity of Parkinsonian manifestations (UPDRS) and cognitive decline (ACE-R) (Table 6B and Figure 9A). Notably, CYC1 mRNA was also significantly increased in the NS/PDS group compared with the HC subjects (P=0.21), but to a lesser extent than in the GZ/PD group (P=0.01) [Figure 8]. The highest levels of CYC1 , as well as FMR1 and ASFMRl transcripts in PBMCs were found in two carriers of the 85 and 170 CGG alleles, respectively (Figures 8A to D). These findings implied, first that the changes in CYC 1 expression as seen in PM/GZ carriers may be relevant to the FMR1 mRNA toxicity, thus contributing to the severity of clinical manifestations of Parkinsonism. Secondly, considering that the CYC1 expression levels are closely linked to the degree of mitochondrial expansion or depletion (Li et al, 1996 supra), the findings here indicated that mitochondrial dysfunction was involved in toxicity of the FMR1 transcripts.

[0120] Therefore, as the next step, the levels of mtDNA were assessed in whole blood, where significantly reduced levels of expression of ND1 gene mitochondrial DNA was found in a sample of PDS carriers (GZ/PDS) compared with both a subset of the NS/PDS (P=0.025) and HC (P=0.019) groups (Figure 8D). Despite the small sample sizes, ND1 mtDNA depletion was also significantly related to the number of CGG repeats within the PDS/GZ category (P=0.01 1) [Figure 9C], and to the rate of cognitive decline represented by MMSE scores (PO.001) [Figure 9D].

[0121] Data indicated (Figure 10) that increased ASFMRl expression (at least in some PDS subjects) may be linked to dopamine deficiency. Although ASFMRl expression was not significantly elevated in the L-dopa untreated PDS/normal size allele group as a whole there was an increasing trend in expression compared to healthy controls and L-dopa treated PD and normal size allele groups. [0122] Data further indicated (Figure 1 1) that PDS carriers of GZ alleles do not respond as well to L-dopa treatment as GZ non carriers, at least in terms of mitochondrial aspects that may be involved in the pathology of the disease. This is evident from: (i) the significantly decreased mtDNA in the treated PDS /GZ carriers compared to treated PDS individuals with normal size CGG alleles; (ii) the mtDNA levels in the L-dopa treated PDS /GZ carriers being equivalent to the mtDNA levels the L-dopa untreated PDS individuals with normal size CGG alleles. [0123] Data presented here show that the presence of either GZ or lower-end PM alleles, may contribute to: (i) the pathogenesis of Parkinsonism through the cytotoxic effect of elevated FMR1 and/or ASFMR1 mRNA involving mitochondrial dysfunction (ii) poor response to dopamine supplementation. This indicates a link between elevated transcription at the FMR locus and CGG expansion within GZ/PM range with mitochondrial dysfunction and this may be more general than just neurodegenerative disorders and may involve dopaminergic system and the related neuropsychiatric conditions.

Table 6

Results of robust regression analysis between neurological and neuropsychological scores, and the molecular measures in the carrier (GZ/PDS), and non carrier (NS/PDS) groups

(A) CGGs FMRl ASFMR1

Variables 1 Estimate s.e. P n Estimate s.e. P 8 Estimate s.e. P

UPDRS M 0.686 0.132 <0.001 li 32.867 13.787 0.032 B 27.013 12.305 0.050

Catsys Tl H 0.013 0.007 0.085 12 -0.292 0.454 0.532 W 3.371 0.429 < 0.0001

Catsys RT B 0.002 0.001 0.013 0.121 0.1 15 0.315 If 0 183 0.082 0.062

ACE-R H -0.212* 0.137* 0.133* 16 -20.305 12.448 0.127 If -20.369 8.367 0.035 MSE -0.070* 0.024* 0.006* iff -3.278 1.885 0.104 -5.120 1. 169 0.001

SMDT 11 -0.393 0.198 0.055 -31.262 15.171 0.060 II -25.733 10.440 0.033

(B) CYC1 ND1

m

Variables n Estimate s.e. P n Estimate s.e. P

UPDRS §> 33.585 8.581 0.002 IS - 10.942 10.716 0.327

Catsys Tl n -0.1 17 0.425 0.787 I T -0.334 0.326 0.332

Catsys RT pi 0.137 0.091 0.160 :1 T -0.067 0.074 0.385

ACE-R 11 -25.775 10.306 0.027 9.1 13 7.085 0.225

MMSE ^•15 -2.576 1 .871 0.192 1 2.788 1.410 0.074

SMDT .15 -26.571 14.876 0.097 11 14.445 10.874 0.21 1

* Measures adjusted for age.

[0124] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

A language and environment for statistical computing [computer program]. Version: R Foundation for statistical computing; 2009

Ayub & Hallett, Aging Cell 3(4): 145-149, 2004

BaskerviUe and Douglas, CNS Neurosci Ther. f3J:e92-123, 2010

Bennet et al, N. Engl. J. Med. 334:71-76, 1996

Berry-Krvais et al, Ann Neurol 53:616-623, 2003

Biniszkiewicz et al, Mol Cell Biol 22:2124-2135, 2002

Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974

Calabresi et al, Trends in Neuroscience 77:4536-4544, 1997

Chen et al, Hum Mol Genet 72:3067-3074, 2003

Conners et al, Psychopharmacol Bull 32:67-73, 1996

Cook, Acta Neurol Scand 752: 109-1 14, 1994

Cousins et al, Biopolar Disord (¾):787-806, 2009

Dahl et al, Clin Chem 53^:790-793, 2007

Fahy e/ al, PCR Methods Appl. l(]):25-33, 1991 Fatemi et al, Schizophr. Res. 2010, doi: 10.1016/j.schres.2010.07.017

Fu et al, Cell 67: 1047- 1058, 1991

Gitan et al, Genome Res. 72^: 158-164, 2002

Godler et al, BMC Clin Pathol 9:5, 2009

Hagerman et al, Neurology 57^: 127- 130, 2001

Hall et al, Parkinsonism and Related Disorders 2010

doi: 10.1016/j .parkreldis.2010. 07.006

Handa et al, FEBS Lett 579, 2702-2708, 2005

Heaton, Wisconsin Card Sorting Test Manual: Revised and expanded (TX:Psychological Assessment Resources, Odessa), 1993

Holden et al, Am J Med Genet (54:313-318, 1996

Irwin et al, Cereb Cortex 70(70j: 1038- 1044, 2000

Jacquemont et al, Am J Ment Retard 109(2): \ 54- \ 64, 2004

Jacquemont et al, JMed Genet 42(2):e l4, 2005

Jin et al, Neuron 39(5):Ί39-Ί4Ί , 2003

Jin and Warren, Hum. Mol. Genet 9^:901-908, 2000

Khalil et al, PLoS ONE. J(7J:el486, 2008 haniani et al, Mol Cytogenet 1:5, 2008

Kukull et al, J. Clin. Epidemiol. 5^: 1050-1058, 1994

Lawson et al, Epilepsia 47: 1540-1545, 2000

Lenth, Java Applets for Power and Sample Size [Computer software],

http://www.stat.uiowa.edu/~rlenth/Power, 2006:2009

Li et al, Eur J Biochem 241 (2):649-656 1996

Loesch et al, Clin Genet 67(5):4 \ 2-4 \ l , 2005

Loesch et al, J Med Genet 44:200-204, 2007

Lord et al, Autism Diagnostic Observation Schedule Los Angeles: Western Psychological Services, 1999

Louis and Luchsigner, Arch Neurol (55:717-722, 2006

Maddalena et al, Genet Med 5:200-205, 2001

Marmur and Doty, J. Mol. Biol. 5: 109, 1962

Mathuranath et al, Neurology 55(11): \ 613- 1620, 2000

Mitchell et al, Clin Genet 67:38-46, 2005

Monte-Silva et al, J Psychol, 2010 Nolin et al, Am JHum Genet 72^:454-464, 2003 Nygren et al, Nucleic Acids Res. 33(14):e\2S, 2005

Penagarikano et al, Ann Rev Genomics and Human Genetics 5: 109-129,2007

Pietrobono et al, Nucleic Acids Res 30(14):3278-32S5, 2002

Pietrobono et al, Hum Mol Genet. 14(2):26Ί-2ΊΊ , 2005

Pieretti et al, Cell 66(4): 17-822, 1991

Primary Care Companion J Clin Psychiatry. 7(2):62-69, 2005

Proitsi et al, Neurobiol Aging, 2010

Richards et al, Neurology 45:2184-2188, 1993

Ross-Inta et al, Biochem J 429:545-552, 2010

Scott et al, Cerebral Cortex 11(10):9\ 8-923, 2001

Smith, Learn Disord 3:83-91 , 1968

Smith, Symbol Digit Modalities Test, Los Angeles, CA, USA, Western Psychological Services, 1991

Sparrow & Cicchetti, Appl Res Ment Retard 5:219-231 , 1984

Spreen & Benton, Neurosensory Center Comprehensive Examination for Aphasia, 1977 Tassone et al, Am J Hum Genet. 66(l):6- \ 5, 2000

Terracciano et al, Am JMed Genet C Semin Med Genet 13 7C( 1 : 32 - 37 ^', 2005 Tost et al, Nucleic Acids Res 3I (9):e50, 2003

Trenerry, Stroop Neuropsychological Screening Test (Psychological Assessment Resources, Florida), 1988 van Os and Kapur, Lancet 22 374(9690):635-45, 2009

Vandesompete et al, Genome Biol 3(7): RESEARCH 0034, 2002

Verkerk et al, Cell 65^ :905-91 4, 1 991

Wechsler (ed), The Wechsler Adult Intelligence Scale-Third Edition: Adminsitration and Scoring Manual, (Orlando: The Psychological Corporation), 1997

Wilson et al, Neurology 58: 1 81 5- 1 81 , 2002

Wojdacz et al, Nucleic Acids Res. 35(6):e4 \ , 2007

Yamouchi and Nagura, Stroke 25:965-969, 1997

Yegnasubramanian et al, Nucleic Acids Res. 34(3): \ 9, 2006

Claims

CLAIMS:

1. An assay to assess the severity of symptoms associated with a neurological condition in a subject, said method comprising determining the level of expression of a genetic region or protein selected from FMR1 , ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein clinical symptoms are considered severe when:

(i) there is an elevation in expression of FMR1 and/or ASFMR1 in whole blood and its constituents or a decrease in expression of FMR1 in Epstein Barr virus (EBV)- transformed lymphocytes in the presence of a CGG expansion associated with a grey zone (GZ) mutation compared to healthy controls;

(ii) there is a decrease expression of FMR1 , mRNA and/or protein in EBV-transformed lymphocytes in the absence of any CGG expansion compared to healthy controls;

(iii) there is an increase in expression of CYC1 or Caspase 8 in the presence of a GZ expansion compared to healthy controls;

(iv) there is a decrease in expression of CYC1 in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls;

(v) there is an increase in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with a neurological condition compared to healthy controls; and/or

(vi) there is an increase in expression of FMR1 sense and/or ASFMR1 antisense transcripts.

2. The assay of Claim 1 wherein the neurological condition is selected from Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and an autism spectrum disorder.

3. The assay of Claim 2 wherein the Parkinsonism is typical or atypical Parkinson's disease, Parkinson's disease spectrum or a Parkinsonian syndrome.

4. The assay of Claim 2 wherein the neurological condition results from mitochrondrial or dopaminergic system dysfunction.

5. The assay of any one of Claims 1 to 4 wherein the subject is a human.

6. An assay to assess the severity of symptoms associated with a neurological condition in a subject, said method comprising determining the level of expression of a genetic region or protein selected from FMR1 , FMRP, ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein clinical symptoms are considered severe when there is an elevation in expression of FMR1 or FMRP and/or ASFMR1 in whole blood and its constituents or a decrease in expression of FMR1 in Epstein Barr virus (EBV)- transformed lymphocytes in the presence of a CGG expansion associated with a grey zone (GZ) mutation compared to patients with a neurological condition with normal size CGG expansion in FMR1.

7. A method for monitoring treatment of a neurological condition in a subject, said method comprising providing the subject with a therapeutic protocol and then determining the level of expression of a genetic region or protein selected from FMR1 , ASFMR1 , CYC1 and Caspase 8 or the genes encoding or associated with mitochondrial complex III or a diagnostic equivalent thereof in cells from the subject wherein the treatment protocol is considered effective when:

(i) there is a decrease in expression of FMR1 and FMRP and/or ASFMR1 in whole blood and its constituents or an increase in expression of FMR1 in EBV-transformed lymphocytes in the presence of a GZ mutation;

(ii) there is an increase in expression of FMR1 in EBV-transformed lymphocytes in the absence of any CGG expansion;

(iii) there is a decrease in expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) there is an increase in expression of CYC 1 in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is a decrease in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with Parkinsonism compared to healthy controls;

(vi) there is an increase in expression of mitochondrial complex III in EBV-transformed lymphocytes in the absence of a GZ expansion; and/or

(vii) there is a decrease in or normalization of expression of FMR1, FMRP and ASFMR1, and mtDNA levels in blood or its constituents.

8. The method of Claim 1 wherein the neurological condition is selected from Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and an autism spectrum disorder.

9. The method of Claim 8 wherein the Parkinsonism is typical or atypical Parkinson's disease, Parkinson's disease spectrum or a Parkinsonian syndrome.

10. The method of Claim 8 wherein the neurological condition results from mitochondrial or dopaminergic dysfunction.

1 1. The method of any one of Claims 7 to 10 wherein the subject is a human.

12. Use of expression of a gene or protein selected from FMR1, FMRP, ASFMR1 , CYC1 , mitochondrial complex III and Caspase 8 or a diagnostic equivalent thereof in the manufacture of an assay to determine the clinical severity of a neurological condition or the efficacy of a therapeutic protocol to a neurological condition.

13. Use of Claim 12 wherein the neurological condition is selected from Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and an autism spectrum disorder.

14. Use of Claim 13 wherein the Parkinsonism is typical or atypical Parkinson's disease, Parkinson's disease spectrum or a Parkinsonian syndrome.

15. Use of Claim 13 wherein the neurological condition results from mitochondrial or dopaminergic dysfunction.

16. Use of any one of Claims 12 to 15 wherein the use is in a human.

17. A kit when used for an assay of any one of Claims 1 to 6.

18. A kit when used for a method of any one of Claims 7 to 1 1.

19. An assay to identify an agent which reduces the severity of symptoms associated with a neurological condition, said method comprising determining the level of expression of a genetic region or protein selected from FMR1 , FMRP, ASFMR1 , CYC1 , mitochondrial complex III and Caspase 8 or a diagnostic equivalent thereof in the presence of an agent to be tested in cells from a test subject wherein an agent is selected when:

(i) there is a decrease in expression of FMR1 and/or FMRP and/or ASFMR1 in whole blood and its constituents or an increase in expression of FMR1 in EBV-transformed lymphocytes in the presence of a GZ mutation;

(ii) there is an increase in expression of FMR1 in EBV-transformed lymphocytes in the absence of any CGG expansion;

(iii) there is a decrease in expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) there is an increase in expression of CYCl in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) there is a decrease in expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with Parkinsonism compared to healthy controls;

(vi) there is an increase in expression of mitochondrial complex III in EBV-transformed lymphocytes in the absence of a GZ expansion; and/or

(vii) there is a decrease in expression of FMR1 and/or ASFMR1.

20. The assay of Claim 19 wherein the agent is further selected if there is improved mitochondrial function and/or improved functioning of the dopaminergic system.

21. The assay of Claim 19 or 20 wherein the neurological condition is selected from Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and an autism spectrum disorder.

22. The assay of Claim 21 wherein the Parkinsonism is typical or atypical Parkinson's disease, Parkinson's disease spectrum or a Parkinsonian syndrome.

23. The assay of Claim 22 wherein the neurological condition results from mitochondrial or dopaminergic dysfunction.

24. Use of a gene or gene product selected from FMR1 , FMRP, ASMFR1 , CYC 1 , mitochondrial complex III and Caspase 8 or an equivalent thereof in the manufacture of a medicament in the treatment of a neurological condition in a subject.

25. Use of Claim 24 wherein the neurological condition is selected from the neurological condition is selected from Parkinsonism, dementia, schizophrenia, bipolar disorder, chronic depression and an autism spectrum disorder.

26. Use of Claim 25 wherein the Parkinsonism is typical or atypical Parkinson's disease, Parkinson's disease spectrum or a Parkinsonian syndrome.

27. Use of Claim 26 wherein the neurological condition results from mitochondria] or dopaminergic dysfunction.

28. An agent identified by the method of any one of Claims 24 to 27.

29. An agent which reduces the severity of symptoms associated with a neurological condition, said method comprising determining the level of expression of a genetic region or protein selected from FMR1 , FMRP, ASFMR1 , CYC1 , mitochondrial complex III and Caspase 8 or a diagnostic equivalent thereof in the presence of an agent to be tested in cells from a test subject wherein an agent:

(i) decreases expression of FMR1 and/or FMRP and/or ASFMR1 in whole blood and its constituents or an increase in expression of FMR1 in EBV-transformed lymphocytes in the presence of a GZ mutation;

(ii) increases expression of FMR1 in EBV-transformed lymphocytes in the absence of any CGG expansion; (iii) decreases expression of CYCl in whole blood and its constituents or Caspase 8 in EBV-transformed lymphocytes in the presence of a GZ expansion;

(iv) increases expression of CYCl in EBV-transformed lymphocytes in the absence of a GZ expansion;

(v) decreases expression of mitochondrial complex III in whole blood and its constituents in the absence of a GZ expansion in patients with Parkinsonism compared to healthy controls;

(vi) increases expression of mitochondrial complex III in EBV-transformed lymphocytes in the absence of a GZ expansion; and/or

(vii) decreases expression of FMR1 and/or ASFMR1.