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Diagnostics - Parkinson’s Disease
Arrayit has leveraged the company’s patented and proprietary microarray discovery platform to decipher the molecular basis of Parkinson’s Disease (PD). This important research project offers exciting promise for the pre-symptomatic and post-symptomatic diagnosis and treatment of this serious neurodegenerative condition. Arrayit is currently seeking a strategic partner to accelerate FDA approval of the Arrayit PDx™ Parkinson’s Diagnostic Test, speed the benchmarking of PDx™ as a companion diagnostic, and determine and enhance the efficacy of pipeline therapeutic proteins and small molecules.
Obama administration proposes new project to map the human brain. Click here for the New York Times article.
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Rationale
Arrayit believes that many and possibly all human illnesses are caused by the improper expression of subsets of cellular genes that alter specific cellular processes, leading to defined disease states. In many cases, mutations in the DNA blueprint alter protein function, which in turn causes elevated or reduced expression of specific genes essential for proper cellular function. This rationale has an important implication: human diseases including Parkinson’s Disease are readily decipherable by quantitative analysis of gene expression at the level of the whole human genome. We used this rationale to decipher PD.
Gene Expression Profiling
Arrayit performs quantitative gene expression analysis of the entire human genome using the company’s H25K Whole Human Genome Chip. H25K is a glass-substrate slide based DNA microarray that contains gene-specific capture elements for each of the 25,509 (25K) genes comprising the human genome. H25K was used to decipher the molecular basis of Parkinson’s Disease using gene expression profiling, a process that measures and compares the activity of each gene to the expression observed in a control sample.
Experimental Approach
Arrayit approached Parkinson’s Disease by examining cell samples from PD and control patients. Patient biopsies were used to establish permanent cell culture lines for Parkinson’s patients containing mutations in one or both copies of the LRRK2 gene, the most common genetic marker for Parkinson’s Disease. In the case of patients with no known family history of Parkinson’s Disease and no identifiable genetic alteration (sporadic PD), patient blood samples provided the source of cellular RNA. RNA was isolated from the cultured cell lines and blood samples, amplified to produce a larger amount of material, converted into labeled cDNA containing fluorescent dye molecules, and hybridized to H25K DNA microarrays to allow whole genome gene expression profiling. H25K microarrays were washed to remove unbound cDNA, scanned to produce fluorescent images and analyzed using Arrayit Genome Pathway Builder™ bioinformatics software to produce a PD gene expression signature. The entire experimental process is complete within 24 hours.
Gene Expression Data
Arrayit H25K DNA microarray data obtained by scanning a fluorescent H25K microarray hybridized with labeled cDNA from a patient with Parkinson’s Disease. The scanned region, which corresponds to an 18 x 54 mm area on the H25K microarray, provides gene expression measurements for 25,509 human genes and 795 controls. The scanning process is complete within 10 minutes.
Parkinson’s Disease Biomarkers
Arrayit identified biomarkers for Parkinson’s Disease by detecting red and green spots in H25K microarray data. The red-green two color labeling method was employed to ensure that all markers identified in the study accurately reflect the PD disease state. In the two-color approach, one H25K microarray (left) was hybridized with a fluorescent probe mixture containing Parkinson’s patient cDNA labeled with red Cy5 dye and the spousal control sample was labeled with a green Cy3 dye. A second H25K microarray (right) was hybridized with a fluorescent probe mixture containing Parkinson’s patient cDNA labeled with green Cy3 dye and the spousal control sample was labeled with a red Cy5 dye. Bona fide biomarkers for PD appear as red spots on the first microarray and green spots in the second microarray. This “dye swap” approach eliminates biomarker false positives and confers a technical advantage for Arrayit in the marketplace.
Parkinson’s Disease Gene Expression Signature
Arrayit H25K analysis of 18 Parkinson’s Disease patients identified a characteristic PD gene expression signature comprising 11 activated genes and 19 repressed genes out of the 25,509 genes in the human genome. PD patients with LRRK2 genetics and sporadic disease exhibited one of three gene expression sub-signatures: (1) 11 activated genes, (2) 11 activated genes and 19 repressed genes, or (3) 19 repressed genes. Patients with one or two copies of the mutated LRRK2 gene produced expression signature 2 and 1 respectively, suggesting that LRRK2 mutations are responsible for/contribute to the observed changes in gene expression. Integer values shown in the table correspond to gene expression ratios in PD test and spousal control samples.
PDx™ Research Test Data
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Patient ID
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Age
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Genotype
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Diagnosis
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PDx™ Result
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AC4061.1
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NA
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Normal
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Healthy
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Negative
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AC4771.1
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60
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LRRK2 -/-
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PD
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Positive
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AC1671.1
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NA
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Normal
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Healthy
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Negative
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AC5771.1
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35
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LRRK2 -/+
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Pre-symptomatic
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Positive
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AC5141.1
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NA
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Normal
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Healthy
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Negative
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AC8451.1
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NA
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Sporadic
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PD
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Positive
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AC4181.1
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69
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Normal
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Healthy
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Negative
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AC6851.1
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71
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LRRK2 -/+
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PD
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Positive
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AC6181.1
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76
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Normal
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Healthy
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Negative
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AC9761.1
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74
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LRRK2 -/+
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PD
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Positive
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AC4061.2
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NA
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Normal
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Healthy
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Negative
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AC4771.2
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60
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LRRK2 -/-
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PD
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Positive
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AC1671.2
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NA
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Normal
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Healthy
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Negative
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AC5771.2
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35
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LRRK2 -/+
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Pre-symptomatic
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Positive
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AC5161.1
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92
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Normal
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Healthy
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Negative
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AC3931.1
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72
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LRRK2 -/+
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PD
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Positive
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AC2181.1
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58
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Normal
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Healthy
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Negative
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AC1851.1
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62
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LRRK2 -/+
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PD
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Positive
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Arrayit PDx™ Parkinson’s Disease Test data for samples with known PD genetics including patient ID, age, PD genotype, diagnosis and PDx™ research test result. NA, age data not available. PDx™ produced negative test results for all patients with normal LRRK2 genetics and a healthy diagnosis for a test specificity of 100%. PDx™ produced positive test results for all patients heterozygous (LRRK2 -/+) or homozygous (LRRK2 -/-) for the p.G2019S LRRK2 allele and positive for Parkinson’s Disease diagnosis (PD) for a test sensitivity of 100%. PDx™ produced a positive test result for two patient samples with LRRK2 mutations but pre-symptomatic for Parkinson’s Disease. Given that age 60 is the average age of onset for Parkinson’s Disease and PDx™ detects PD markers in individuals as early as age 35, the data show that PDx™ provides a 25 year pre-symptomatic window for drug therapy to treat and cure Parkinson’s Disease. The company believes that PDx™ is the only research test available that offers 100% specificity, 100% sensitivity and a 25-year pre-symptomatic detection window for PD.
PDx™ Test Advantages
Arrayit PDx™ Parkinson’s Disease Test has key advantages over other markers and marker panels described previously. One key advantage is that PDx™ measures patient gene activity in real time by quantifying mRNA levels (the products of cellular genes), rather than identifying hardwired mutations in the DNA code such as the p.G2019S LRRK2 marker through genotyping that may or may not correlate with Parkinson’s Disease. The disconnect between genotyping and PD is caused by compensatory mutations (“second site suppressors”) in the genome that override the genetic consequences of mutations such as LRRK2, which explains why some LRRK2 individuals are lifetime asymptomatic whereas others have full-blown Parkinson’s Disease. PDx™ is 100% accurate (no false positives or false negatives) because the test accounts for the complexities of PD genetics. Another PDx™ advantage is that the test allows pre-symptomatic detection in patients as early as 25 years before the appearance of symptoms rather than scoring for pleiotropic markers that appear only in late-stage symptomatic patients. All of the pre-symptomatic patients that have scored positive with PDx™ to date have one or more senior family members with full-blown PD, reinforcing that fact that PDx™ correctly detects PD in pre-symptomatic individuals. PDx™ also provides definitive binary “yes-no” test results rather than probabilistic information based on likelihoods and percentages of acquiring PD. The company believes that these advantages confer Arrayit a significant competitive advantage in the diagnostics and therapeutics marketplaces.
Parkinson’s Disease Bioinformatics
Arrayit analyzed 31,500,000 H25K gene expression data points from 18 patients with Parkinson’s Disease using Arrayit Genome Pathway Builder™ (GPB) software, which utilized multivariate calculus, fuzzy logic and artificial intelligence to construct a biological pathway for Parkinson’s Disease. Biological pathway construction was helpful because it provided a molecular basis for PD and will allow a targeted approach to the discovery and testing of therapeutic proteins and drugs including mesencephalic astrocyte-derived neurotrophic factor (MANF).
Parkinson’s Disease Biological Pathway
Arrayit bioinformatic analysis of 18 Parkinson’s patients using Genome Pathway Builder™ (GPB) software allowed us to construct a best-fit biological pathway for Parkinson’s Disease. The modeled pathway responsible for PD symptoms appears to involve a cellular malfunction in the pathway that mediates protein folding, misfolding and removal inside human cells. This model is provocative because it is consistent with the neuropathology observed in Parkinson’s patients whereby accumulated protein aggregates disrupt the function of the dopaminergic cells comprising the substantia nigra. The promising protein therapeutic lead compound mesencephalic astrocyte-derived neurotrophic factor (MANF) appears to work by exerting its mode of action on the same biological pathway.
Parkinson’s Disease Therapeutics
Several companies are developing small molecule and protein therapeutics for Parkinsons’ Disease. The Arrayit PDx™ Test provides a molecular assay for benchmarking and optimizing PD medicines and targeting treatment to bona fide PD patients.
FDA Approval
Arrayit PDx™ Pre-Symptomatic Test for Parkinson’s Disease will be submitted to the United Sates Food and Drug Administration (FDA) as a 510(k) submission in the near future.
Copyright 1993-2013 Arrayit Corporation. All rights reserved.
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