The results of this study support our hypothesis that there are blood gene biomarkers that can distinguish early PD patients from normal control subjects. Notably, 38 out of the 62 Parkinson cases in the mild/early cohort were de novo and so, not treated with any antiparkinsonism drug when the blood samples were obtained while the rest were collected during the first year of medication. This suggests that the genetic signature could be an early diagnostic marker for PD. In support, the classifier model performed equally well in early stage de novo PD samples, producing a similar ROC AUC value to that obtained with the entire early PD cohort (de novo and medicated), indicating that patient medication had no significant effect on the PP of the classifier for PD risk and that the model is stable throughout the two PD groups. Supporting this concept, it was recently shown in a population of asymptomatic LRRK2 mutation carriers, that reduced CSF amyloid β and tau species correlated with lower striatal dopaminergic function as determined by PET , suggesting that they may serve as potential biomarkers even in asymptomatic phases of the disease. The performance of the gene model was validated in an independent cohort of patients at advanced PD stage where all individuals were correctly classified as PD, while it fully discriminated PD from a group of individuals affected with AD (considered the most common neurodegenerative disease). Giving that misdiagnosis occurs normally at the initial PD stage, the 100% sensitivity obtained with the long-term PD cohort support the feasibility of the biomarker panel to differentiate with certainty between PD and non-PD. Further studies will determine the ability of the panel to differentially diagnose idiopathic PD from patients with other forms of Parkinsonism, such as PSP and MSA.
One main challenge in the development of biological markers is to minimize the number of genes in the classification model while still achieving a high classification rate. The present biomarker signature identified a minimal set of transcripts in blood that has a high discriminating power to categorize the PD early group and to positively/negatively classify the advanced PD and AD cohorts.
A model with fewer genes is likely to yield better generalization (less number of free variables) and optimization of diagnosis. We have found that five out of the seven gene transcripts previously reported to have been changed in sporadic PDSN , were found altered in blood of mild/early PD. Our findings argue in support of the view that changes in peripheral blood may have relevance to mechanisms occurring in brain of PD patients and indicate that at least some of the gene expression alterations occurring in PD are not exclusive to the brain, but are expressed also in peripheral blood tissue. Indeed, a large proportion of the genes encoded in the human genome have detectable levels of transcripts in circulating blood cells ; When coming into contact with brain tissue, circulating blood cells may provide information concerning the pathological environment of the PD brain.
Gene expression correlation analysis indicates a significant association in blood from healthy control individuals between SKP1A and five gene transcripts: HIP2, ALDH1A1
HSPA8 and EGLN1, while it was absent in early PD, suggesting a functional coordinative role for Skp1. Skp1 takes part in the ubiquitin-proteasome/E3-ligase SCF complex, acting in a module-like manner: Skp1 can interact with several F-box proteins, which play an indispensable role in the selection of target proteins for degradation . Thus, a reduced activity of Skp1 may play a role in the development of PD by impairing the timely degradation of a wide array of proteins, promote their deposition and affect the function of dopaminergic (DAergic) neurons. Skp1, together with the chaperone Hsc-70 encoded by HSPA8, the proteasomal ATPase subunit PSMC4, the EGLN1-encoded prolyl hydroxylase and the huntingtin-interacting protein Hip2, are intimately connected to processing/degradation of proteins by UPS/lysosomal- mediated degradation [17–19, 34–37]. Further evidence for a possible functional connection between the panel genes is provided by our recent finding showing that silencing SKP1A in the SN-derived murine cell line SN4741 induced a parallel down-regulation in the transcripts of ALDH1A1 and HSPA8. Aldh1 was found to be expressed highly and specifically in DA cells of the SN and ventral tegmental area (VTA)  having a role in the neutralization of toxic aldehyde derivatives of DA . These highly reactive, neurotoxic aldehydes can accumulate in case of decreased levels of Aldh1, as occurs in SNpc of PD [39, 40], and can promote neuronal death. The fact that the five genes comprising the signature, as a group, play important roles in PD neuropathology and are significantly correlated in blood form healthy subjects, add a biological significance to the findings.
Supporting the rationale of identifying molecular changes in peripheral blood that may respond to the pathology in the brain of sporadic PD, Grunblatt et al.  recently reported a cluster of four genes in blood tissue that discriminated between PD and healthy controls. One of them, ALDH1A1 was also detected in our gene signature, independently confirming part of our results. Further support comes from Scherzer et al.  who demonstrated a panel of eight genes involved in relevant PD processes such as the UPS, mitochondrial function and apoptosis in whole blood tissue from a heterogeneous cohort of relatively early-staged PD individuals, that correlated with PD risk. It is worth noting that despite the difference in the study design, e.g., the use of large-scale microarrays comprising the whole genome, the restricted eight-gene signature included HIP2, also found by us, as a surrogate for PD. In our study, we have performed multi-step logistic regression analysis, which is commonly applied in biomarker research. This procedure recruits in each step the most significant gene discriminating between PD and control in relation to the prior step, thus taking into consideration the cumulative impact of the gene group on the PD risk. In Scherzer’s study, the genes were individually rank-ordered according to the absolute value of their correlation coefficient with PD, disregarding the correlation between their expression levels.
Another major discovery of this investigation is that the PP values of the five-gene signature were accentuated in patients at late PD stage, suggesting a potential for the model to assess disease severity. One relevant point is what could be the biological meaning of this observation. It can be conjectured that the peripheral gene transcriptional changes may reflect evolution of pathogenic processes during PD progression. In analogy, Shi et al.  have described a panel of seven CSF proteins that could aid in PD diagnosis and differential diagnosis. Among these, an increase in CSF fractalkine, along with decreased Aβ1–42 levels, correlated with a higher UPDRS score in cross-sectional samples and in a set of longitudinally collected PD samples from the DATATOP study.
When examining the relative quantity of each gene individually at the cross-sectional level, we demonstrated a similar transcriptional pattern for SKP1A, ALDH1A, PSMC4 and HSPA8 in the two PD cohorts compared to normal controls or AD groups, suggesting that these transcripts are altered at early stages of the disease and not affected by disease progression. However, at this stage, we cannot determine whether the selective elevation of HIP2 demonstrated only in PD patients at advanced stage of disease, can reflect a disease evolution. Despite the strength of the present findings, there are some issues yet to be addressed. At this point the cross-sectional nature of this study does not allow making a correlation between gene expression and clinical symptoms that may point to the clinical state. Longitudinal studies will establish whether the gene panel can serve as a marker for PD risk or its progression. Although we have initially focused on seven out of the 20 gene transcripts most altered in sporadic PD brains, it is likely that the other risk genes could be also relevant.