1.4.1.2.1 Huntington’s Disease
Huntington’s disease (HD), which has a prevalence of 12.3 per 100,000 of the UK Caucasianpopulation(Pringsheim et al 2012),isan adult onset movement disorder of monogenic inheritance that is caused by a polyQ expansion of 40 repeat lengths or greater in exon 1 of the HTT gene encoding the huntingtin protein (MacDonald et al 1993). The condition is characterised by the loss of gamma-Aminobutyric acid or GABAergic neurons and widespread reactive gliosis in the striatum and cerebral cortex (Runne et al 2007). Extrapyramidal signs include rigidity and bradykinesia, dystonia, reduced coordination, weight loss and chorea with cognitive decline and psychiatric disturbances (e.g. irritability, anxiety & depression) normally apparent before the onset of overt motor impairment (Ross & Tabrizi 2011).
Transcriptionalprofilingcomparingpre-symptomatic(n=5)orsymptomatic(n=12) HD patients to fourteen unrelated, age and gender matched, neurologically normal healthy control subjects identified 322 of ~11,000 probes (2.9%) common to both
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Affymetrix Human Genome U133A GeneChip® Arrays and Amersham BioSciences
CodeLink Uniset Human I and II bioarrays with an average expression ratio of >1.8 or<0.6(student’st-testp<0.0005)forupand down-regulatedchanges,respectively. The majority of transcripts were increased in peripheral blood of patients relative to controls and reflected diverse biological processes including transcription/RNA processing, ubiquitin-mediated proteasomal degradation, vesicular trafficking and signaltransduction. Thetop30highestranking genes, as determined by probability valueandfold-change,wereassessedbyqRT-PCRand a subset of 12 demonstrating themostsignificantDEselectedtoformabiomarker panel which could be applied, usingprincipalcomponentanalysis(PCA)mappingsoftware,todistinguishnotonly between HD and controls, but also between different stages of disease progression with pre-symptomatic patients clustering distinctly between individuals which are symptomatic and those that are neurologically healthy in two independent cohorts comprising a) 16 late pre-symptomatic, 14 symptomatic and 25 controls or b) nine early pre-symptomatic and a further nine controls. The panel also proved effective in a Phase I clinical trial in monitoring individual patient responses to the histone deacetylase (HDAC) inhibitor sodium phenylbutyrate (Borovecki et al 2005) which has been shown to exhibit neuroprotective properties in a transgenic mouse model of the disease (Gardian et al 2005). Moreover, an up-regulation in the expression of 7 of the 12 candidate genes (58.3%) was confirmed by qRT-PCR in human derived post-mortem tissue from the caudate nucleus of five additional HD brains and four controls (Borovecki et al 2005). In another study comparing HD (n=61), PD (n=20) andischemicstroke(n=10)patients,thesameapproachwasconductedbyLovrecic et al (2009) using a standard logistic regression based model which was successful ingeneratingapositivepredictivescoreof78%withsensitivity 82% and specificity 53%. This finding, however,could not be replicated in lymphocytes suggesting that this particular panel of biomarkers may be specific to blood (Runne et al 2007).
1.4.1.2.2 Parkinson’s Disease
PD affects approximately 1% of the population over the age of 65 (Wirdefeldt et al 2011) and is characterised as a slowly progressive neurodegenerative disorder that is characterised by a resting tremor, rigidity, gait abnormalities, asymmetric
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bradykinesia and postural instability.Pathologicallytheconditionishallmarkedby the presence of α-synuclein containing LB’s as well as a substantial loss of dopaminergic neurons from within the substantia nigra pars compacta region of the brain (Ali & Morris 2014).
Transcriptionalprofiling of 50 early stage PD patients with a mean Hoehn and Yahr scoreof2.3(range1-4)and55unrelatedcontrolsincluding33diseasemimics[ALZ, progressive supranuclear palsy (PSP), CBS or multiple systems atrophy (MSA)] and 22neurologically healthy control subjects which were age, gender and blood count matched identified 22 unique genes represented on the Affymetrix Human Genome U133A GeneChip® Arrays as being DE between PD patients and controls with SAM
(significance analysis of microarrays) FDR p<0.03 and a fold-change (FC) threshold of ±1.25. Functional categories that were highlighted included the cell cycle, DNA/ RNA turnover, protein phosphorylation & transport, carbohydrate metabolism and apoptosis.Ofparticularinterestisthe down-regulation of the heat shock protein 70 (HSP70) co-chaperone suppression of tumorigenicity 13, ST13 (⇩1.63-fold, p<0.01) whichhasbeenimplicatedintheaberrant folding of α-synuclein and its subsequent toxicity towards dopaminergic neurones in PD (Flower et al 2005, Klucken et al 2004).Thiswaslaterconfirmedintwo independent qRT-PCR assays using different housekeeping genes (Scherzer et al 2007).Theauthorsalsodividedthecohortinto atrainingset(62.9%) [31 PD, 18 mimics and 17 controls] and a test set (37.1%) [19 PD, 15 mimics and 5 controls] in order to determine an optimum biomarker panel of 8 candidate genes that correlated significantly with the risk of developing PD [OR of third tertile leave one out cross-validation 5.7 with 95% confidence interval (CI) between 1.6 and 21, p<0.01] (Scherzer et al 2007). In a re-analysis of the aforementioned dataset, Soreq et al (2008) were able to further improve the discriminativepowerofthestudy by deploying distribution plots and PCA mapping to allow for the effective removal of outliers that may have arisen from technical inconsistencies. In doing so they have highlighted previously unreported changes in the mRNA transcript levels of genes relating to neuro-immune signalling (Soreq et al 2008). It is also worth noting that an intron 8 SNP encoded within one of these markers,VDR(vitaminDreceptor),is found to be disproportionately over-represen tedinKoreanPDsufferersrelative to the general population (Butler et al 2001, Kim
48 et al 2005, Li et al 2014).
In a more recently published report by Grunblatt et al (2010), 4 of 12 (33.3%) pre- selected candidates based upon prior GEP analyses of human post-mortem derived brain tissue [ALDH1A1, HIST1H3, LAMB2 and PSMA2] (Grunblatt et al 2004, Hauser et al 2006, Lu et al 2005, Simunovic et al 2009, Zhang et al 2005) could successfully differentiate between sporadic PD cases [11 drug naïve and 116 mediated], disease mimicsdiagnosedwithALZandneurologicallyhealthy,elderlycontrolsubjectswith a sensitivity and specificity of greater than 80% in qRT-PCR assays of RNA isolated from peripheral whole blood (Grunblatt et al 2010).
1.4.1.2.3 Alzheimer’s Disease
ALZrepresentsthemostcommonformofage-relateddementiathatafflictsbetween 3 and 7% of the global population older than 75 years (Abdulrahman & Jnr 2014, Takizawa et al 2014).Progressivememorylossandadeclineincognitivefunctionis accompanied by reactive gliosis, atrophy of the basal forebrain and hippocampus, extracellular depositions of Aβ (senile plaques) and intracellular accumulations of hyperphosphorylated tau (neurofibrillary tangles) (Duyckaerts et al 2009, Sabuncu et al 2011).
Transcriptional profiling of peripheral blood mononuclear cells (PBMC’s) from 14 mildly symptomatic sporadic ALZ patients [Mini Mental State Examination (MMSE) scoresof23.4±3outofapossible30]andanequalnumberof neurologically healthy age and education matched elderly control subjects using the National Institute on Aging(NIA) Human Mammalian Gene Collection (MGC) cDNA Arrays identified 942 of 6,424 probes as being DE including 849 (90.1%) down-regulated and 93 (9.9%) up-regulated transcripts with an average expression ratio of >1.2 or <0.9 and SAM 2-wayanalysisofvariance(ANOVA)FDR threshold of p<0.05. Gene Set Enrichment Analysis (GSEA) using the online open source database that is freely available from the GeneExpression Omnibus (GEO) repository highlighted genes associated with apoptosis,proteintranslation&inflammationthat were significantly increased and genes associated with cytoskeletal maintenance, cellular trafficking, mitochondrial function, lipid metabolism, redox homeostasis, neurotransmission, transcription &
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DNA repair or cellular response to stress which were significantly decreased in the PBMC’s of ALZ patients in relation to the controls (Maes et al 2007). Whilst a large proportion of these have been found to be gender specific, changes in 78% of those selected in males and 56% in females were confirmed in a qRT-PCR assay (albeit in the same cohort of samples used in the microarray experiments) and >160 probes exhibited similar expression changes in human derived post-mortem brain tissue (Blalock et al 2004, Colangelo et al 2002, Yao et al 2003) and/or transgenic animal models of the disease (Dickey et al 2003, Ho et al 2001, Reddy et al 2004).
1.4.1.2.4 Schizophrenia and Bipolar Disorder
Schizophrenia(SCHIZ)andBipolardisorder(BPD)areseverelydebilitating chronic remitting and relapsing neuropsychiatricillnessesthateachoccurin approximately 1% of the population. The former is characterised by psychosis with symptoms of hallucinations & delusions, disorganised speech, catatonia and variable degrees of motor, cognitive and/or social dysfunction (Tandon et al 2013, Tandon et al 2009); whereas the latter, conversely, is a mood affective disorder that is characterised by episodes of mania and depression and is often associated with paranoia as well as sleep-wake disturbances (Fagiolini et al 2013, Oswald et al 2007).
Transcriptional profiling of individuals of Han Chinese descent including SCHIZ (n= 30)andBPD(n=16)patientsdiagnosedaccordingtotheIVcriteriaofthe Diagnostic and Statistical Manual of Mental Disorders (DSM) and 28 unrelated, neurologically healthy control subjects identified 89 of 12,674 probes (~0.7%) on the Affymetrix U133A/plus2.0 GeneChip® Arrays as being DE between the two patient groupings
and controls, applying a non-parametric ANOVA (Kruskal-Wallis) p<0.005 without performing a multiple comparisons correction. Two-dimensional (2D) hierarchical clusteringusingtheSpearman’scorrelationstatisticdemonstratedthreegenetically defined subgroups on the HeatMap which corresponded to SCHIZ, BPD and control cases in the original microarray experiment. Logistic regression and ROC (Receiver Operating Characteristic) curve analysis from pair-wise comparisons of SCHIZvctrl, BPDvctrl and SCHIZvBPD determined linear and non-linear combinations of eight ofthehighestranking genes, which were confirmed by qRT-PCR, to be successful in discriminating not only between a healthy versus disease status but also between
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SCHIZandBPDwithanoverall efficiency of 95% (Tsuang et al 2005). Many of these putative markers were found to reside at chromosomal loci which have previously been linked to SCHIZ [1cen.q12 (ADSS), 1q21 (S100A9), 20q13 (DATF1) and 22q13 (APOBEC3B)], BPD [4p16 (CTBP1) and 4q21 (CXCL1)] or alternatively both of these neuropsychiatric conditions [11q22 (ATM) and 19q13 (CLC)] (Badner & Gershon 2002, Cassidy et al 2007, Cheng et al 2006, Francks et al 2010, Fullerton et al 2010, Lewis et al 2003). Furthermore, an increase in the expression levels of chemokine (C-X-C motif) ligand 1 melanoma growth stimulating activity, alpha (CXCL1) (⇧1.58 -fold, p<0.001) has been independentlyverifiedinasecondqRT-PCR cohort which was comprised of an additional 30 SCHIZ patients and 26 unrelated, neurologically healthy control subjects (Yao et al 2008).