CA3071824A1 - Biomarkers associated with parkinson's disease - Google Patents

Abstract

Disclosed herein are biomarkers related to the identification or diagnosis of Parkinson's disease in a subject. The disclosed biomarkers and related methods are used to detect or predict Parkinson's disease status in a subject. The disclosed biomarkers and methods can be utilized to determine nature of Parkinson's disease, progression, or responsiveness to a treatment or prophylaxis. Some embodiments include methods of detecting the presence of a biomarker associated with a patient. Some embodiments of the methods include generating a report for clinical disease evaluation or management.
Some embodiments of the compositions include associated kits, peptides, binding members, and uses thereof. Also provided are methods and compositions for diagnosing, preventing, and treating Parkinson's disease in a subject in need thereof.

Description

BIOMARKERS ASSOCIATED WITH PARKINSON'S DISEASE
Related Applications This application claims the benefit of U.S. Provisional Application No.
62/539,620, filed on August 1, 2017 and U.S. Provisional Application No. 62/598,423, filed on December 13, 2017. The entire teachings of the above applications are incorporated herein by reference.
Technical Field of Invention The technical field of this invention relates to medicine and cell biology of the human body. Various embodiments relate to methods and compositions involving biomarkers for the diagnosis, treatment, and prevention of Parkinson's disease. The present invention is further directed to biomarkers of Parkinson's disease and to the use of these compounds in identifying a subject suspected of having or being susceptible to Parkinson's disease. The present invention is further directed to associated kits, peptides, binding members and uses thereof Summary of the Invention Surprisingly, the inventors have discovered novel polypeptid.e biomarkers, referred to herein as "Parkinson Associated Factors (PAFs)", that are associated with Parkinson's disease. In particular, the presence of one or more PAFs in a biological sample of an individual corresponds to a positive Parkinson's disease status or an increased risk of Parkinson's disease. Therefore, PAFs are useful as a biomarker of Parkinson's disease.
The present invention provides novel polypeptide biomarkers designated Parkinson Associated Factor (PAFs). Various embodiments relate generally to methods and compositions involving PAF-1, PAF-2, PAF-3, PAF-4 that comprise one or more polypeptides having an amino acid sequence comprising 12 amino acid residues set for in SEQ ID NOS: 1-4. Various embodiments relate generally to methods and compositions involving a precursor PAF that comprises a carboxy-terminal CaaX motive having an amino acid sequence set for as CTIA or CVIA. Various embodiments relate generally to methods and compositions involving a PAF that comprises a prenylation, such as famesylation or geranylgeranylation of the C-terminal cysteine residue.
Preferably, a PAF
comprises farnesylation of the C-terminal cysteine residue. Uses of compounds of the present invention to treat, ameliorate, diagnose, or prevent Parkinson's disease are disclosed. Uses of compounds of the present invention to promote treatment of Parkinson's disease are disclosed. Uses of compounds of the present invention in combination with clinical diagnostics and/or radiographic medical imaging to treat, ameliorate, diagnose, stage, or prevent Parkinson's disease are disclosed.
Provided herein are methods of assessing a Parkinson's disease status in an individual. Also provided herein are methods of assessing a Parkinson's disease risk status in a biological sample of an individual. Preferably, methods comprise the steps of obtaining a sample from the individual; obtaining a biomarker level comprising Parkinson Associated Factor (PAF) in the sample; comparing the biomarker level from the individual to a reference biomarker level corresponding to a known Parkinson's disease status; and categorizing the individual as having the known Parkinson's disease status if the biomarker level information of the individual does not differ significantly from the reference biomarker level information. Various aspects of these methods are recited below, contemplated as distinct or in combination.
Methods are also contemplated to include methods wherein obtaining a biological sample comprises a biological sample collection tool such as collecting from the individual an undershirt or other clothing item that has been worn by the individual.
Methods are also contemplated to include methods wherein the biomarker information also includes Parkinson disease risk factor information for the individual, such as, e.g., age, family history, genetic forms, and the like.
Methods are also contemplated to comprise transmitting a report of results of an assay that uses on or more PAFs as a diagnostic and/or prognostic marker to a health practitioner or other desirable entity. Optionally, the report recommends that a treatment for Parkinson's disease be implemented or considered. Optionally, the individual undergoes a Parkinson's disease treatment. Optionally, the report recommends undergoing an assay or a clinical evaluation for diseases associated with increased risk in individuals with Parkinson's disease, such as, e.g., melanoma, seborrheic dermatitis, neurogenic orthostatic hypotension, pseudobulbar affect, and the like. Optionally, the individual undergoes an assay or a clinical evaluation for diseases associated with increased risk in individuals with Parkinson's disease. Optionally, the report recommends administering an anti-Parkinson's disease composition. Optionally, an anti-Parkinson's disease composition is administered to the individual. Optionally, the report recommends continued monitoring of Parkinson's disease status. Optionally, the obtaining of a biomarker level comprises contacting a fraction of the biological sample to a binding member, such as an antibody or

2 set of antibodies, wherein the antibody or antibodies comprises an antibody or antibodies specific to a PAF. Optionally, determining in a sample of biological fluid obtained from the subject the presence or amount of an anti PAF antibody. Optionally, comparing the presence or amount of the anti PAF antibody with a pre-defined threshold value and assigning a diagnosis of Parkinson's disease or a future likelihood of developing Parkinson's disease when the presence or amount of an antibody against PAF is detected or exceeds the threshold. Optionally, the obtaining PAF level comprises subjecting a fraction of the biological sample to a mass spectrometric analysis.
Optionally, the obtaining PAF level comprises subjecting a fraction of the biological sample to a gas chromatographic analysis. Optionally, at least one of the comparing and the categorizing is performed on a computer configured to analyze reference biomarker information.

Biomarker levels disclosed herein distinguish samples having a chromatographic reference compound (CRC) signal not only from samples from healthy individuals but also from samples from individuals having other types of neurodegenerative disorders or diseases.
Examples of other types of neurodegenerative disorders or disease include 1-methyl-4-pheny1-1,2,3,6-tetrahydroppidine (MPTP), rotenone, manganese, and annonacin exposure induced neurotoxicities.
Also provided herein are methods of monitoring efficacy of a Parkinson's disease treatment regimen in an individual. Some such methods comprise the steps of obtaining a first sample comprising a biological substance from the individual at a first time point;
administering the treatment regimen to the individual; obtaining a second sample comprising a biological substance from the individual at a second time point;
obtaining a first biomarker level comprising PAF level in the first sample and a second biomarker level comprising PAF level in the second sample; wherein a change in biomarker levels indicates efficacy of the Parkinson's disease treatment. Parkinson's disease treatment regimens are contemplated herein and are known to one of skill in the art, such as pharmacological intervention, e.g., levodopa therapy; administration of a biologic therapeutic agent, e.g., embryonic stem cells; and surgical intervention, e.g., deep brain stimulation (DBS).
Also provided herein are ex vivo methods of monitoring efficacy of a Parkinson's disease treatment in an individual. Some such methods comprise the steps of obtaining a first sample comprising a biological substance from the individual at a first time point;
obtaining a second sample comprising biological substance from the same individual receiving a Parkinson's disease treatment at a second time point; obtaining a first

3 biomarker level comprising PAF levels in the first sample and a second biomarker level comprising PAF levels in the second sample; wherein a change in biomarker levels indicates efficacy of the Parkinson's disease treatment.
Various aspects of these methods are recited below, contemplated as distinct or in combination.
Methods are contemplated to include obtaining a sample that comprises drawing blood from a vein or artery of the individual, cerebrospinal fluid from a spinal tap of an individual, and gaseous constituents of a closed space over skin of an individual.
Optionally, the Parkinson's disease treatment or treatment regimen comprises administration of a pharmaceutical composition. Optionally, the Parkinson's disease treatment or treatment regimen comprises administration of an antifungal agent.
Methods are also contemplated to include methods comprising comparing the first biomarker level and the second biomarker level to at least one biomarker level of a healthy reference, wherein the second biomarker level being more similar to the biomarker level of the healthy reference indicates efficacy of the Parkinson's disease treatment.
Optionally, methods comprise the first biomarker level and the biomarker level to at least one biomarker level of a healthy reference, wherein the first biomarker level being more similar to the biomarker level of the Parkinson's disease reference indicates efficacy of the Parkinson's disease treatment. Optionally, methods comprise changing the Parkinson's disease treatment or treatment regimen if no efficacy is indicated.
Optionally, methods comprise repeating Parkinson's disease treatment or the treatment regimen if no efficacy is indicated. Optionally, methods comprise continuing the Parkinson's disease treatment or treatment regimen if no efficacy is indicated. Optionally, methods comprise discontinuing the Parkinson's disease treatment or treatment regimen if efficacy is indicated.
Also provided herein is a biomarker indicative of a Parkinson's disease status of an individual in need thereof.
Also contemplated herein is a biomarker for use in assessing a Parkinson's disease status according to any of the above methods or monitoring efficacy of a Parkinson's disease treatment according to any of the above methods.
Also provided herein is a kit or kits, comprising an antibody or set of antibodies that identifies PAF. Various aspects of the kits are recited below, contemplated as distinct or in combination. Kits are contemplated to comprise an antibody that binds to a control protein. Optionally, kits comprise an antibody or antibodies to PAF and another non-PAF
antigen. Optionally, the antibody kit comprises antibodies that identify other biomarkers

4 of disease. Optionally kits comprise instructions functionally related to use of the kit to assess a Parkinson's disease status of an individual in need thereof. Also contemplated herein are kits for use in assessing a Parkinson's disease status according to any of the above methods or monitoring efficacy of a Parkinson's disease treatment according to any of the above methods.
Also contemplated herein are computer systems configured to assess a Parkinson's disease risk in an individual in need thereof. Some such computer systems comprise a memory unit for receiving data comprising measurement of a biomarker comprising PAF
from a biological sample comprising gaseous constituents of a closed space above skin of an individual, computer-executable instructions for assessing a Parkinson's disease risk associated with the measurement of the biomarker, an output unit for delivering a report assessing the Parkinson's disease risk associated with the measurement of the biomarker.
Optionally, the memory unit is configured for receiving data comprising measurement of a second biomarker. Optionally, the data comprising measurement of a biomarker comprises an immunohistochemical analysis result such as enzyme-linked immune-sorbent assay (ELISA) data. Optionally, the data comprising measurement of a biomarker comprises mass spectrometry data. Optionally, assessing a Parkinson's disease risk comprises comparing the data to a reference biomarker data associated with a known Parkinson's disease status. Optionally, the individual is assigned the known Parkinson's disease status when the data does not differ significantly from the reference biomarker data.
Optionally, the reference biomarker data indicates presence of Parkinson's disease.
Optionally, the reference biomarker indicates absence of Parkinson's disease. Optionally, assessing a Parkinson's disease risk is performed on a computer configured to analyze reference biomarker information. Optionally, the memory unit comprises at least one reference .. biomarker information set corresponding to a known Parkinson's disease status.
Optionally, the at least one reference biomarker information set comprises a machine teaming model. Optionally, the report recommends that a treatment be performed.
Optionally, the report recommends an independent intervention. Optionally, the report recommends undergoing an independent assay or clinical evaluation. Optionally, the report recommends undergoing a melanoma cancer screening assay. Optionally, the report recommends undergoing a seborrheic dermatitis screening assay. Optionally, the report recommends undergoing an odor identification screening assay. Optionally, the report recommends administering an anti-Parkinson's disease composition. Optionally, the report includes a recommendation of continued surveillance of health status of the individual.

5

6 Computer systems herein are also contemplated wherein at least one parameter of the individual's reference biomarker information differs significantly from a corresponding value from the reference biomarker information set, and wherein the individual's reference -biomarker information does not differ significantly from the reference bionaarker information set. Optionally, the memory unit is configured to receive Parkinson's disease risk factor information from the individual. Optionally, the computer-executable instructions factor in one or more risk factors (e.g., age, family history) of the individual when assessing the Parkinson's disease risk associated with the measurement of the biomarker.
Biomarker accumulation levels are measured in a number of ways in various embodiments, for example through an ELISA assay, through mass spectroscopy analysis or through alternate approaches to protein accumulation level quantification.
Mass spectrometric techniques typically utilized in biomarker based clinical diagnosis, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrotnetry; (LC-MS), ambient ionization mass spectrometry (also referred to as "ambient mass spectrometry"), electrospray ionization ES! MS, tandem MS
(MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF/MS), are contemplated. Contemplated herein are various sample pretreatment methods which improve the detection efficiency of disease bionaarkers as well known in the art Due to an ability to rapidly detect large biomolecules in trace amounts, MALDI-TOF/MS is a preferred tool for characterizing disease biomarkers in biologic samples.
Biomarker accumulation levels are compared to a positive control or negative control standard, or to a model of Parkinson's disease accumulation levels or of healthy accumulation levels, such that a prediction is made regarding an assayed health status of an individual in need thereof. In some cases, a biomarker assay result is accompanied by a recommendation regarding an intervention or an alternate verification of the biomarker assay results.
The invention also provides methods of identifying at least one biomarker PAF
protein in a patient for diagnosing a patient with Parkinson's disease or a patient at risk for developing Parkinson's disease comprising the steps of, obtaining a biological sample from the patient; measuring the level of PAF protein in the sample; and optionally comparing the level of PAF protein from the patient to a control sample obtained from an individual whose Parkinson's disease status is known. Preferably the at least one biomarker PAF protein is selected from SEQ ID NOS: 1-4, optionally wherein the terminal cysteine of any one of SEQ ID NOS: 1-4 is independently a C-terminal geranylgeranylcysteine comprising an attached carboxymethyl group or a C-terminal farnesylcysteine comprising an attached carboxymethyl group. Preferably, measuring the level of PAF protein comprises determining the amount of biomarker PAF protein in the .. biological sample using mass spectrometry (MS) or immunoassay analysis or both.
Preferably, the MS analysis comprises matrix assister laser desorption/ionization (MALDI) time of flight (TOF) MS analysis or electrospray ionization (ES!) MS, tandem mass spectrometry (MS/MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Preferably the immunoassay analysis .. comprises an enzyme-linked immunosorbent assay (ELISA).
The invention also provides methods of detecting the presence or absence of at least one biomarker PAF protein selected from PAF-1, PAF-2, PAF-3 or PAF-4 in a patient comprising, assaying a biological sample obtained from the patient for the presence or absence of PAF-1, PAF-2, PAF-3 or PAF-4. Preferably assaying the biological sample comprises contacting the biological sample with antibodies specific for PAF-1, PAF-2, PAF-3 or PAF-4, and detecting the presence or absence of antibodies bound to PAF-1, PAF-2, PAF-3 or PAF-4. Preferably, assaying the biological sample comprises using mass spectroscopy (MS) to detect the presence or absence of PAF-1, PAF-2, PAF-3 or PAF-4 in the biological sample.
Provided herein are biomarkers and assays useful for the diagnosis and/or treatment of at least one of Parkinson's disease.
Also provided herein are kits, comprising a computer readable medium, and instructions for use thereof.
Incorporation By Reference All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Detailed Description of the Invention For convenience, certain terms employed in the entire application (including specification, examples, and appended claims) are defined throughout the specification.
Unless defined otherwise, all technical and scientific terms used herein have the same

7 meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
While the specification concludes with claims that particularly point out and distinctly claim the invention, it is believed the present invention will be better understood from the following description. The present invention may comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well any of the additional or optional ingredients, components, or limitations described herein.
All parts, percentages, and ratios are based upon the total weight of the compositions of the present invention, unless otherwise specified. All such weights as they pertain to listed compounds are based on the compound level and, therefore; do not include carriers or by-products that may be included in formulations of listed compounds.
The components and/or steps, including those, which may optionally be added, of the various embodiments of the present invention, are described in detail below.
All documents cited are, in relevant part, incorporated herein by reference;
the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.
Except as otherwise noted, all amounts including quantities, percentages, portions, and proportions, are understood to be modified by the word "about", and amounts are not intended to indicate significant digits.
Except as otherwise noted, the articles "a", "an", and "the" mean "one or more".
Except as otherwise noted, the article "or" means "or", "and", and "and/or".
As used herein, the term "comprising" means that other steps and other ingredients that do not affect the end result can be added. The term "comprising"
encompasses the terms "consisting of and "consisting essentially of. The compositions and methods/processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.
Within the present invention it is to be understood that the combinations, compositions or combined uses according to this invention may envisage the simultaneous, sequential or separate use of the steps, components, or compositions. In this context, "combination" or "combined" within the meaning of the present invention may include, without being limited, fixed and non-fixed (e.g. free) forms (including kits) and

8 uses, such as e.g. the simultaneous, sequential or separate use of the steps, components, or compositions.
As used herein, the term "effective" means an amount of a substance high enough to provide useful (e.g., medically actionable) information or to provide a significant positive modification of a condition to be treated or protected from. The term "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be measured in one or more measurements or administered in one or more administrations. In the context of treatment and protection, an "effective amount" is that amount sufficient to ameliorate, stabilize, reverse, slow or delay progression of the target infection or disease states. An effective amount of the substance will vary with the particular condition being treated or detected, the severity of the condition, the duration of treatment, the nature of concurrent treatment, and like factors.
As used herein, the term "efficacy" means the degree to which an amount of a substance is effective.
As used herein, the terms "treat", "treating, "ameliorate" and "ameliorating"
are used interchangeably herein with respect to Parkinson's disease, and mean to improve, reduce, mitigate, prevent (e.g., prophylaxis), reverse (e.g., alleviate), control or manage the signs or symptoms associated with Parkinson's disease and further to making the subjective or objective measures of quality or quantity of life better for patients at risk of or suffering from Parkinson's disease.
As used herein, the term "differential level" of a biomarker may include any increased or decreased level. In one embodiment, differential level means a level that is decreased by: at least 5%; by at least 10%; by at least 20%; by at least 30%;
by at least 40%; by at least 51%; by at least 60%; by at least 70%; by at least 80%; by at least 90%;
by at least 100% (i.e., indicative of the absence of the biomarker molecule).
In another embodiment, differential level means a level that is increased by: at least 5%; by at least 10%; by at least 20%; by at least 30%; by at least 40%; by at least 51%; by at least 60%;
by at least 70%; by at least 80%; by at least 90%; by at least 100%; by at least 110%; by at least 120%; by at least 130%; by at least 140%; by at least 150%; by at least 200%; or more.
As used herein, the term "biological sample" refers to any preparation from a cell, tissue, fluid (e.g., secretion), or volatile emission (e.g., breath) of a subject. Preparations include, without limitation, serum, plasma, cells of tissues, fluids, gases, and the like. A
biological sample includes any substance obtained from the body of the subject, such as,

9 without limitation, biological fluids such as urine, saliva, perspiration, and blood, including derivatives of blood, e.g. plasma, and serum, and biological gases such as breath. Examples of bodily substances include, without limitation, intravascular fluid, interstitial fluid, lymphatic fluid, transcellular fluid, radial artery puncture fluid, venipuncture fluid, amniotic fluid, aqueous humour, vitreous humour, bile, blood, blood serum, blood plasma, mammal), gland secretions, cerebrospinal fluid, otis cerumen, chyle, chyme, gingival crevicular fluid, endolymph, perilymph, perspiration, feces, vaginal secretions, gastric secretions, nasal exudates, sinus exudates mucus, pericardial fluid, peritoneal fluid, pleural fluid, serous fluid, suppurative exudate, emesis, flatuence, mucopurulent discharge, rheum, saliva, sebum, semen, smegma, sputum, synovial fluid, lacrimal secretions, urine. A "skin sample" refers to a biological sample comprising a preparation from a skin or epidermis of a subject, including, e.g., gaseous constituents emanating and vaporous emissions from the skin or epidermis.
As used herein, the term a "biomarker" refers to a substance whose level of expression or presence in the bodily sample obtained from the subject experiencing or predisposed to experiencing an undesirable physiological state (e.g., Parkinson's disease) differs as compared to the biological sample obtained from a subject not experiencing or not predisposed to experiencing an undesirable physiological state (e.g., control or "healthy" subject). The term "predisposed" refers to the increased likelihood of the subject having Parkinson's disease when compared to the subject's physiological state in the absence of any indication of Parkinson's disease.
The term "invasive sampling" refers to invasive methods for isolating a biological sample includes the use of needles, for example during blood sampling, as well as biopsies of various tissues (e.g., curettage), blistering, or photoporation collection techniques. In contrast, "non-invasive" sampling refers to any procedure that does not require insertion of a device or instrument through a body orifice or skin for collecting a biological sample (e.g., for use in diagnosis or treatment of a subject in need thereof).
The term "objectively" means without bias or prejudice. In contrast, the term "subjectively" refers to any self-generated or expert assessments or expression of opinion.
The term "baseline" means information gathered at the beginning of a study from which variations found in the study are later measured. A baseline sample may be taken from a healthy or diseased subject. A reference measurement gathered at the beginning of a previous study or studies may also provide baseline information.

As used herein, the terms "polypeptide", "polypeptide fragment", "protein", and "peptide" are used interchangeably to refer to an optionally isolated polymer of amino acid residues composed of linearly arranged amino acids linked by peptide bonds.
The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, isoprenylation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component. Proteins may comprise chains of amino acids linked by amide bonds between the carboxyl and amino groups of adjacent amino acids.
Proteins are not limited to a minimum length unless otherwise defined. A peptide is a protein that typically contains between at least two amino acid residues to less than about 50 amino acids in length. Proteins may be produced biologically and isolated from the natural environment, produced synthetically (typically using naturally occurring amino acids) (e.g. chemical peptide synthesis), or produced using a recombinant technology (e.g., cell-free protein synthesis (CFPS)). In some aspects, the polypeptide or protein is a "modified polypeptide" comprising non-naturally occurring amino acids. In some aspects, the polypeptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids. In some aspects of all the embodiments of the invention, the peptides or modified peptides further comprise co-translational and post-translational (e.g., C-terminal peptide cleavage) modifications, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, prenylation (e.g., famesylation, geranylgeranylation), lipidation, methylation, selenocystine modification, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, proteolysis (e.g., metalloprotease cleavage), and the like.
The term "geranylgeranylation" refers to the attachment of a 20-carbon lipophilic geranylgeranyl isoprene unit to a cysteine amino acid residue located at the C-terminus of a protein. The geranyl-geranyl group is attached through a thioether bond to a cysteine residue. The term "geranylgeranylcysteine" (GG-Cys) refers to a cysteine amino acid residue with an attached geranylgeranyl group. Geranylgeranylation is a form of prenylation.

The term -famesylation" refers to the addition of a famesyl group to peptides or proteins bearing a CaaX motif (i.e., a four-amino acid sequence at the carboxyl terminus of the peptide or protein). The famesyl group is a 15-carbon isoprenoid lipid.
Famesylation is a type of prenylation. Prenylation (also referred to as isoprenylation or lipidation) is a post-translational modification of proteins by which hydrophobic molecules, such as an isoprenyl group, are added to a protein or chemical compound.
In some aspects of the invention, the polypeptide is altered or modified which includes alterations, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity or property. These modifications can be deliberate, as through site-directed mutagenesis (e.g., CRISPR/Cas9 editing), or can be accidental, such as through mutations of artificial hosts, such as genetically engineered bacteria, yeast or mammalian cells, that produce the proteins, or errors due to recombinant DNA methods (e.g., PCR amplification). In some embodiments, a polypeptide of the present invention is fused to at least one heterologous polypeptide. In some further embodiments, the heterologous protein is an Fc polypeptide. In yet further embodiments, the Fc polypeptide is attached via a linker sequence. Variant or alternative forms of the biomarker include for example polypeptides encoded by any splice-variants of transcripts encoding the disclosed biomarkers. In certain cases, the modified forms, fragments, or their corresponding RNA
or DNA, may exhibit better discriminatory power in diagnosis than the full-length protein.
Biomarkers contemplated herein also include truncated forms or polypeptide fragments of any of the proteins described herein. Truncated forms or polypeptide fragments of a protein can include C-terminally deleted or truncated forms and N-terminally deleted or truncated forms. Truncated forms or fragments of a protein can include fragments arising by any mechanism, such as, without limitation, by alternative translation, exo-and/or endo- proteolysis and/or degradation, for example, by chemical, physical, and/or enzymatic proteolysis.
All amino acid residues identified or described herein are in natural or L-configuration unless otherwise specified. Typical peptide nomenclature abbreviates commonly described amino acids as follows:
G - GUY - glycine F - PHE - L-phenylalanine M - MET - L-methionine A - ALA - L-alanine S ¨ SER ¨ L-serine I ¨ ILE ¨ L-isoleucine L ¨ LEU ¨ L-leucine T ¨ THR ¨ L-threonine V ¨ VAL ¨ L-valine P ¨ PRO ¨ L-proline K ¨ LYS ¨ L-lysine N ¨ ASN ¨ L-asparagine H ¨ HIS ¨ L-histidine Q ¨ GLN ¨ L-glutamine E ¨ GLU ¨ glutamic acid W ¨ TRP ¨ L-tryptophan R ¨ Arg ¨ L-arginine D ¨ ASP ¨ L-aspartic acid C ¨ CYS ¨ L-cysteine.
As used herein, the term "percent sequence identity" refers to the degree (e.g., 50%, 600/0, 70%, 80%, 90%, 95%, 98%, 99%, etc.) to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. If two polymers have identical sequences (e.g., 1000/0 sequence identity) they may be referred to herein as having sequence identity. As used herein, the term "percent sequence similarity" refers to the degree (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, etc.) with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics or can be grouped according to conservative amino acid substitution properties. A "conservative" amino acid substitution refers to the substitution of an. amino acid in a polypeptide with another amino acid having similar properties, such as size or charge. In certain embodiments, a polypeptide comprising a conservative amino acid substitution maintains at least one activity of the unsubstituted polypeptide. A conservative amino acid substitution may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties.
Naturally occurring residues may be divided into classes based on common side chain properties, for example: hydrophobic: methionine, alanine, valine, leucine, norleucine, and isoleucine; neutral hydrophilic: cysteine, serine, threonine, asparagine, and glutamine;
acidic: aspartic acid and glutamic acid; basic: histidine, lysine, and arginine; residues that influence chain orientation: glycine and proline; and aromatic: tryptophan, tyrosine, and phenylalanine. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class; whereas conservative substitutions may involve the exchange of a member of one of these classes for another member of that same class. If two polymers have sequences that have monomers at each position that share the same biophysical characteristics they may be referred to herein as having "sequence similarity." The "percent sequence identity" (or "percent sequence similarity") is based on a calculation that comprises the steps of: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides EXAMPLE-1 and EXAMPLE-2 are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide EXAMPLE-1 and peptide EXAMPLE-have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide EXAMPLE-1 and peptide EXAMPLE-2 would have 100% sequence similarity. As another example, if peptide EXAMPLE-3 is 20 amino acids in length and peptide EXAMPLE-4 is 15 amino acids in length, and 14 out of 15 amino acids in peptide EXAMPLE-4 are identical to those of a portion of peptide EXAMPLE-3, then peptides EXAMPLE-3 and EXAMPLE-4 have 70% sequence identity, but peptide EXAMPLE-4 has 93.3% sequence identity to an optimal comparison window of peptide EXAMPLE-3. For the purpose of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gaps in aligned sequences are treated as mismatches at that position.
The term "isolated polypeptide" or "purified polypeptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the polypeptide is purified to any degree relative to its naturally-obtainable state. A purified polypeptide therefore also refers to a poly-peptide that is free from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. A peptide or polypeptide composition can be "of at least" a certain degree of purity if the polypeptide or peptide is at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or more pure on a weight-to-weight basis.
The term "positive Parkinson's disease status" refers to the condition of an individual having the disease called Parkinson's disease (also called Parkinson disease).
"Parkinson's disease" refers to a chronic, progressive neurodegenerative disorder characterized by any combination of four cardinal signs of disease:
bradykinesia, postural instability, resting tremor, and rigidity (e.g., cogwheel rigidity). Other symptoms of Parkinson's disease include motor dysfunctions that include one or more of bradykinesia, rest tremor (such as pill-rolling tremor), rigidity (e.g., cogwheel rigidity), postural instability, gait difficult (e.g., shuffling gait), hypomimia (masked facies), hypokinetic dysarthria, hypophonia, palilalia, dysphagia, sialorrhea, decreased spontaneous eye blink rate, eyelid opening apraxia, hypometric saccades, impaired vestibuloocular reflex, impaired upward gaze and convergence, respiratory distress, micrographia, incontinence, restless legs syndrome, sleep apnea, dystonia, myoclonus, forward-flexed posture, camptocormia (bent spine syndrome), Pisa syndrome, k-yphosis, scoliosis, psychomotor retardation, freezing (motor block), and festination. Parkinson's disease patients may also suffer from cognitive and/or sensory dysfunction or impairments that include one or more of the following non-limiting examples of dysfunction or impairments:
subcortical dementia, Lewy body dementia, psychomotor retardation, memory difficulty, learning and executive dysfunction (e.g., attention deficit disorder), language impairment, altered personality, impulse control dysfunction (e.g. obsessive behaviors), psychosis, hallucinations, delusions, depression, anxiety, social withdrawal, abulia, sleep disturbances (e.g., insomnia), fatigue, nausea, visual-spatial disturbances, blurred vision, visual contrast insensitivity, vision loss, autonomic dysfunction, olfactory dysfunction (e.g., anosmia, reduced odor detection or discrimination), gastrointestinal dysfunction (e.g., constipation), sensoiy pain, sensory disturbances, dermatological dysfunction (e.g., seborrhea), sebon-heic dermatitis (e.g., dandruff). and rhinon-hea.
Prior to the present invention, Parkinson's disease is a disease solely made by clinical diagnosis, relying exclusively on clinical acumen of a physician (typically a .. neurologist) to identify a subject as having the disease (i.e., positive disease status) or not (i.e., negative status). Epidemiologically speaking, advancing age is a risk factor for Parkinson's disease as, for example, Parkinson's disease is generally considered to have an incidence that increases with age from about 1 percent of individuals age 60 to about 5 percent of individuals age 85 worldwide. An individual may have a positive Parkinson's disease status that is pre-clinical, sub-clinical, silent, or latent defined as a reduced number of viable dopaminergic neurons in the substantia nigra pars compacta of the midbrain of the individual without clinically apparent signs and symptoms of Parkinson's disease.
Loss of smell (anosmia) or loss of odor discrimination is a typical early or presenting symptom of Parkinson's disease.
The term "parkinsonism" or "Parkinsonism" refers to any disease that shares one or more of the signs or symptoms as Parkinson's disease but is distinguished by demonstrating a poor or absent response to anti-parkinson pharmacologic therapies, the presence of non-Parkinson symptomatology or absence of certain Parkinson's disease signs and symptoms, and/or having an identifiable environmental cause (e.g., soursop soup consumption, pawpaw fruit tea, exposure to MPTP, handling agricultural pesticide rotenone, and welding-related manganese exposure) or familial cause (e.g., mitochondrial DJ-1 gene (PARK7) mutations).
As used herein, the term "antigen" refers to a molecule that triggers an immune response by the immune system of a subject, e.g., the production of an antibody or activation of the cellular arm of the immune system, such as activation of phagocytes, natural killer cells, antigen-specific cytotoxic T-lymphocytes, and cytokine modulation.
Antigens can be exogenous, endogenous, or autogenous. Exogenous antigens are those that have entered the body from outside through inhalation, ingestion or injection.
Endogenous antigens are those that have been generated within previously-normal cells as a result of normal cell metabolism, or because of viral or intracellular bacterial infection.
Autogenous antigens are those that are normal protein or protein complex present in the host body but can stimulate an immune response.
Polypeptides of PAF

A PAF polypeptide in accordance with the invention is the 12 amino acid length polypeptide amino acid sequence as set forth in SEQ ID NO: 1 shown below.
YPMGPPVWGTTC (SEQ ID NO: 1).
As used herein, the C-terminal cysteine of SEQ ID NO: 1 refers to the cysteine amino acid residue located at position 12 of the sequence.
Exemplary PAF protein biomarker comprises the full length molecule of the polypeptide sequence of SEQ ID NO: 1, as well as uniquely identifiable fragments of the polypeptide sequence of SEQ ID NO: 1. The biomarker can be, but does not need to be, full length to be informative. In many cases, so long as a fragment is uniquely identifiable as being derived from or representing a polypeptide of SEQ ID NO: 1, it is informative for purposes herein.
Additional polypeptides of PAF consist of the 12 amino acid length polypeptide amino acid sequences as set forth in SEQ IDS NOs: 2 ¨ 4 shown below.
YPLGPPVWGTYC (SEQ ID NO: 2) YPRCPVGWGQTC (SEQ ID NO: 3) YPYCTPGWGQTC (SEQ ID NO: 4) Preferably, the PAF comprises a SEQ ID NO: 1, 2, 3, or 4 wherein the C-terminal cysteine is substituted at the side chain thiol with a hydrophobic group, such as a geranylgeranyl group (referred to herein as a "C-terminal geranylgeranylcysteine") or farnesyl group (referred to herein as a "C-terminal farnesylcysteine") and substituted at the cysteine carboxy with an alkyl, such as a lower alkyl, or, preferably, a methyl group, to make an ester (referred to herein as a "carboxymethyl").
Most preferably, PAF comprises a SEQ ID No: 1, 2, 3, or 4 that has a C-terminal farnesylcysteine containing an attached carboxymethyl group instead of a C-terminal cysteine.
The PAF may also consist of the polypeptide comprising the 12 amino acid length polypeptide amino acid sequence as set forth in SEQ ID NO: 1 (i.e., YPMGPPVWGTTC) that has a C-terminal geranylgeranylcysteine containing an attached carboxymethyl group instead of a C-terminal cysteine.
Provided herein are biomarkers, methods, compositions, kits, and systems for the assessment of health status of a subject, for example through the detection of Parkinson's disease status. Biomarkers, methods, compositions, kits, and systems described herein are used to determine a likelihood that a subject has Parkinson's disease or is at increased risk of having Parkinson's disease through the assay of a biological sample taken from the subject. In some embodiments that biomarker comprises the novel peptide marker Parkinson Associated Factor (PAF) and the non-protein biomarker of age of the individual providing the sample.
Biomarkers as disclosed herein share a property that sensitive, specific conclusions .. regarding an individual's Parkinson's disease status are made using protein level information derived from biological samples (e.g., blood), alone or in combination with other information such as an individual's age, genotypic sex, hereditary/family history, genetic information, health history or other characteristics. A benefit of the present peptide biomarker is that PAF provides Parkinson's disease health status or Parkinson's disease risk assessment, such that a commercially and medicinally relevant degree of confidence (such as sensitivity, specificity or sensitivity and specificity) is achieved using an easily obtained biological sample or samples. Prior to the instant invention, disclosed herein, there is no objective test (such as a brain scan or blood assay) to make a definitive diagnosis of Parkinson's disease. The present invention obviates the need to rely solely upon data obtained from subjective, error-prone clinical assessment, such as a clinician taking a medical history and performing a neurological examination whereby diagnosis of Parkinson's disease rests solely on the ability of the clinician to recognize characteristic Parkinson's disease signs and associated symptoms (also referred to as "Parkinson's disease diagnostic clinical evaluation"). Widespread use of PAF as a Parkinson's disease .. biomarker will improve accurate, early diagnosis rates of Parkinson's disease, and Parkinson's disease health issues that are more easily recognized early in the disease progression, so that the health issues may be more efficiently treated.
Ultimately, effects of the benefit of early and accurate diagnosis of Parkinson's disease or risk of Parkinson's disease may be measured in population-wide reduced morbidity/mortality of disease, and is substantial.
Biomarkers disclosed herein comprise biological molecules that are associated with a positive Parkinson's disease status or increased risk of a positive Parkinson's disease status in an individual.
Readily available information such as individual's age, genotypic sex, weight, height, body mass index or other easily measured or obtained information is also eligible as a marker in some cases. In particular, some biomarker information herein rely upon age, genotypic sex, or age and genotypic sex, as biomarkers.

Common to many biomarkers described herein is the ease with which the biomarkers are assayed in an individual. Biomarkers of the present invention may be readily obtained, e.g., by drawing blood from the subject.
A benefit of the ease with which a biomarker based on collecting gaseous constituents of a closed space above skin of an individual is that invasive assays such as drawing blood from a vein or artery of the individual is not required for biomarker measurement. Similarly, cerebrospinal fluid is not required for biomarker determination.
As a result, biomarker information as disclosed herein may be readily obtained through collection of gaseous constituents of a closed space above skin of an individual in combination with a visit to a doctor's office. In one embodiment, a biological sample is a gaseous constituents sample drawn from a closed space above skin of an individual. The gaseous constituents sample can be a headspace sample comprising volatile and/or semi-volatile molecules contained within the closed space above the skin of an individual.
Methods of static headspace sampling, typically used for the determination of volatile and semi-volatile analytes in liquids and solid matrices, are well known in the art and may be prepared by methods of headspace sample preparation well known to those skilled in the art. Sampling according to this embodiment provides a determination of Parkinson's disease status from a biological sample with a sensitivity and a specificity that renders the outcome of the test reliable enough to be medically actionable. Compliance rates are accordingly substantially higher than are compliance rates for invasive biological sample collection procedures. Exemplary biomarkers disclosed herein comprise proteins or fragments thereof that are recognizably or uniquely mapped to their parent protein.
Biomarkers disclosed herein may be measured through a number of approaches such as an immunological interaction, such as that which occurs in an ELISA
assay through which a protein or protein fragment in a biological sample from an individual are bound to a specific antibody or antibodies, and the extent of binding is quantified as a measure of protein abundance in the sample. ELISA assays capable of measuring biomarker as disclosed herein are contemplated as embodiments of the present disclosure as kits. In some instances, an acquired microfluidic biological sample is contacted with a reagent such that antibodies contained within the reagent bind to a peptide biomarker contained within the biological sample. A biomarker detection device may determine levels of the biomarker and provide comparison to reference values against standardized reference levels for the biomarker contained within the biological sample in order to detect or monitor the status or risk of Parkinson's disease. The biomarker detection device may provide results of the analysis to a healthcare provider or other entity and results may be transmitted (e.g., wirelessly) to an external device or source.
Alternately or in combination, biomarkers are measured through mass spectrometric methods such as MS, MS/MS, MALDI-TOF, ESI-MS or other mass spectrometric approaches. Often, the MS approach quantifies a fragment of a biomarker rather than the full-length protein. However, such approaches are sufficient to determine the protein level of the biomarker to an accuracy sufficient for a Parkinson's disease status assessment as disclosed herein.
Gas chromatography is a well-known technique for fractionating and determining the relative amounts of various components in a biological sample containing a mixture of compounds of differing physical characteristics (e.g., volatility). For example, the sample is vaporized and the entire resulting quantity of gases is passed through an analytical chromatography column. Chromatographic processes such as gas chromatography can rapidly determine the volatiles content of a multi-component biological sample, such as would be present in a biological sample obtained from a human subject.
Once an expression level for a biomarker is determined, a Parkinson's disease status assessment is available for the individual from which the sample is obtained. A
number of approaches are available to one of skill in the art to generate or come to a Parkinson's disease status assessment from an individual's biomarker expression level.
Some assessments rely upon comparison of a biomarker level of an individual to a reference biomarker level, such as a reference biomarker level from an individual known or independently verified to have a negative Parkinson's disease status (e.g., good health), or from an individual known or independently verified to have a positive Parkinson's disease status, such as is the case for an individual having advanced Parkinson's disease.
Alternately a biomarker level of an individual is compared to a reference level constructed from a plurality of individuals of common known Parkinson's disease status. In some cases, the reference is an average of known biomarker levels from a plurality of individuals, or alternately is a range defined by the range of biomarkers levels observed in the reference individuals. A range reference biomarker level is in some cases a weighted range, such that outlier values among the individuals having a common Parkinson's health status are given lower predictive value than biomarkers levels that are common to a plurality or majority or all of the biomarker levels.
In more complex assessment approaches, an individual's biomarker level is compared to a reference level constructed from a larger number of individuals of common known positive Parkinson's disease status, such as at least 5, at least 10, at least 50, at least 100, at least 500 or more individuals. Often, the reference individuals are evenly distributed in health status between positive and negative for a Parkinson's disease status.
Assessment comprises in some cases iterative or simultaneous comparison of an individual's biomarker level to a plurality of references of known health status.
Alternately or in combination, a plurality of known reference biomarker levels are used to train a computational assessment algorithm such as a machine learning model such that a single comparison between a biomarker level of an individual and a reference provides an outcome that integrates or aggregates information from a large number of individuals of common known Parkinson's disease status, such as at least 10, at least 50, at least 100, at least 500, at least 1000 or more individuals. Generation of such a reference often facilitates much faster or more efficient (e.g., using of less computational power) assessment of an Parkinson's disease status of an individual.
A reference is generated from a plurality of reference individual biomarker levels through any of a number of computational approaches known to one of skill in the art.
Machine learning models are readily constructed, e.g., using any number of statistical programming languages or data mining and analytics software such as C++, Python, Lasergene, Mathematica, and Matlab. The present inventions contemplates machine learning models that utilize software that acts as a bridge between an operating system or a database and one or more application, typically on a network system.
An individual's biomarker level is compared to a reference as described herein or otherwise performed by one of skill in the art, and an output assessment is generated. A
number of output assessments are consistent with the disclosure herein. Output assessments comprise a single assessment, typically narrowed by a sensitivity, specificity or sensitivity and specificity parameter, indicating a Parkinson's disease status assessment.
Alternately or in combination, additional parameters are provided, such as an odds ratio indicative of the relative increase in chance of suffering from or increased risk of a Parkinson's disease issue in light of the biomarker level of the individual or bioinarker level assessment.
Results are variously provided to the individual or to a health care professional or other professional or entity. Results are optionally accompanied by a heath recommendation, such as a recommendation to confirm or independently assess a Parkinson's disease status assessment, for example using a clinical assessment of a physical neurological examination performed by a physician clinician.

A recommendation optionally includes information relevant to a treatment regimen, such as information indicating that a treatment regimen such as a polypectorny, radiotherapy, chemotherapy, antibody therapy, biosimilar treatment or other treatment regimen, such as information indicative of success or efficacy of the regimen.
Efficacy of a regimen is assessed in some cases by comparison of an individual's biomarker level at a first time point, optionally prior to a treatment and a later second time point, optionally subsequent to a treatment instance. Biomarker levels are compared to one another, each to a reference, or otherwise assessed so as to determine whether a treatment regimen demonstrates efficacy such that it should be continued, increased, replaced with an alternate regimen or discontinued because of its success in addressing the Parkinson's disease. Some assessments rely upon comparison of an individual's bit-nnarker level at multiple time points, such as at least one time point prior to a treatment and at least one time point following a treatment. Bioniarker levels are compared to at least one reference biomarker level.
The biomarkers, methods, compositions, and kits described herein provide assays for determining Parkinson's disease status or Parkinson's disease risk status based on detection or measurement of a biomarker in a biological sample obtained from a subject.
The present invention further provides an improved method for odor identification training in a dog useful in the detection of Parkinson's disease in a person, wherein said improved method for training comprises the use of a positive identifier of Parkinson's disease which is a farnesylated synthetic peptide corresponding to a Malassezia species A
factor (PAF). Current methods of training dogs to detect Parkinson's disease by odor detection are very time consuming and vary in precision. For example, currently dogs are trained using only t-shirts from people with known Parkinson's disease. A
major drawback here is that it is inconvenient to collect the t-shirts from individuals for dog training. Another major drawback is that Parkinson's disease diagnosis is subjective (via clinical evaluation by the neurologist) and thus there can be false positives such that the t-shirt used for training may be a false positive.
Parkinson's disease may be present as a very early stage disease, such as in a clinically asymptomatic stage, or at a later stage wherein symptoms of the disease are evident. The method of the invention provides odor identification training in dogs using one or several synthesized PAF farnesylated peptides of the invention that correspond to a Malassezia species A factor. The dog trained by the method of the invention allows detection of Parkinson's disease on an article of clothing worn by an asymptomatic or symptomatic diseased person, such as on a t-shirt of that person. Synthesized PAF
farnesylated peptides of the method include, but are not limited to peptides found in Table 1.
Table 1 Peptide Name Species Sequence PAF-1; M.globosa Tyr-Pro-Met-Gly-Pro-Pro-Val-Trp-Gly-Thr-Thr-Cys-(farnesyl) SEQ ID NO: 1 PAF-2; M.restricta Tyr-Pro-Leu-Gly-Pro-Pro-Val-Trp-Gly-Thr-Tyr-Cys-(farnesyl) SEQ ID NO: 2 PAF-3; M. obtusa Tyr-Pro-Arg-Cys-Pro-Val-Gly-Trp-Gly-Gln-Thr-Cys-(farnesyl) SEQ ID NO: 3 PAF-4; M. furfur Tyr-Pro-Tyr-Cys-Thr-Pro-Gly-Trp-Gly-Gln-Thr-Cys-(farnesyl) SEQ ID NO:4 Preferably, the invention provides a method of training dogs in odor detection of Parkinson's disease, comprising training a dog to detect a farnesylated synthetic peptide corresponding to a Malassezia species A factor when present on a surface.
Preferably the farnesylated synthetic peptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ
ID NO: 4. Most preferably, the farnesylated synthetic peptide is SEQ ID NO: 1.
Preferably, the dogs are trained to detect a farnesylated synthetic peptide on a surface such as an article of clothing.
Examples.
Canine Odor Identification Assay.
The purpose of this assay was to confirm expected results of detection of SEQ
ID
NO: 1 by Parkinson Alert Dogs (PAD) dogs, who have been trained to identify an odor that is present on T-shirts worn by individuals suffering from Parkinson's disease.
II. Protocol - Materials and Methods = Dogs: Various breeds of dogs were professionally trained to distinguish T-shirts worn overnight by individuals suffering from Parkinson's disease from t-shirts worn by healthy, Non-Parkinson's disease, subjects.

= Sterile water: Ultra Pure Water, DEPC Treated, 100m1, DNAse, RNAse &
protease free.
= Q-tip: Cotton Tip Applicator 6 inch Sterile.
= Gloves: Nitrile Exam Gloves - Medical Grade, Sterile, Powder & Latex Free.
= Thermos: Thermos (brand) Stainless King 24 Ounce Food Jar, Stainless Steel.
= Pipetter: Eppendorf (brand) Single-Channel Research Plus Adjustable-Volume Pipetters.
= SEQ ID NO: I: Tyr-Pro-Met-Gly-Pro-Pro-Val-Trp-Gly-Thr-Thr-Cys-(farnesyl).
The amino acid sequence of SEQ ID NO: 1 corresponds to Malassezia globosa.
A one milligram (1 mg) quantity of SEQ ID NO: I sample was synthesized by the company GenScript (genscript.com). The SEQ ID NO: I sample was stored at negative twenty degrees Celsius (-20 C) as a lyophilized powder prior to use.
Sample t-shirts were prepared as follows: (i) place four unused cotton t-shirts into four thermoses. (ii) swab one of the t-shirts with a sterile q-tip applicator that has been dipped in a solution composed of 1 milligram of SEQ ID NO: I and approximately 2.5 milliliters of sterile ultra pure water; (iii) close and cap the thermoses;
(iv) double-blind the thermoses identification numbers on thermos labels, (v) thermoses transported to canine odor identification testing facility.
II. Test conditions and set up Sample preparation was conducted in a laboratory setting using sterile technique.
T-shirt odor testing was conducted at the Parkinson's Alert Dogs (PAD) testing facility located at the San Juan Fairgrounds, Friday Harbor, Washington.
III. Testing The PAD Director and professional dog handler acted as the operator. The operator stated the dogs are being run in order of best qualified first, to least qualified going last.
The test was conducted following the same protocol as used when the dogs ran on PD
patient t-shirts. There were 3 controls in which each of three thermoses contained only at-shirt. There was 1 active in which the thermos contained a t-shirt + SEQ ID
NO: 1 (in the regular test this would be a t-shirt worn by a PD patient for 24 hours). The dogs were not rewarded with any treat for locating SEQ ID NO: 1. A trained dog considered a t-shirt to be a "positive hit" when the dog makes an indication while situated in front of said t-shirt of to the operator.
IV. Results TEST 1 - SEQ ID NO: I (found): Dog visited each of the four thermos containers moving in a counterclockwise direction. Dog started with the active thermos and finished with the active thermos. The dog demonstrated a positive hit on the active thermos.
TEST 2 - SEQ ID NO: I (found): Dog visits each container going clockwise. Last container was the active. The dog demonstrated a positive hit on the active thermos.
TEST 3 - SEQ ID NO: I (found): Dog started on active and traveled counterclockwise. The dog appeared to double back after going from active to control but continued in a counter clockwise direction, passed the active and hit on the first subsequence control. The dog then spun apparently looking for the smell in the corner of the room zoning back in toward the active. Dog stopped at the active and looks to operator. According to the operator the dog appeared to be fringing at thermos at location 3 before stopping at the active thermos (at location 4). Fringing means that the dog was looking for the "edge" of the odor. This action of the dog was consistent with the fact that this was the third test, therefore, the prior dog or dogs had disturbed odor.
The dog gave a brief positive hit on the control at location 3. Ultimately the dog demonstrated a positive hit on the active thermos.
TEST 4 - SEQ ID NO: I (not found): The dog traveled anti-clockwise and did not give any indication of knowing the location. The dog looked at the controller on each attempt. Hits on a control at location 1. This dog was on anti-epilepsy medication and other medications (known to causes loss of smell ability in dogs) that had resulted in the dog consistently hitting on the control at location 1 (closest to the entrance of the dog) regardless of the placement of thermoses. This dog was allowed to participate, even though the dog was known to have smell impairment, in order to allow the owner to participate (since she was a volunteer at the organization). Therefore, the operator voided this null test result.

TEST 5 - SEQ ID NO: l (found): The dog moved from bottom right (active) to top right and back to bottom right (active). Then to bottom left. The dog looks at the operator twice and heads past top left to top right, to bottom right (active). The dog looks at operator twice. The dog hit on the active, and the operator ended test. The dog demonstrated a positive hit on the active thermos.
TEST 6 - SEQ ID NO: I (found): The dog started moving on bottom right (active), then counter clockwise to top right. Back to bottom right (active). The dog then looked at the operator, and moved to bottom left and indicates. The dog continued to top left, then top right. The dog looked at the operator. On to the bottom right (active).
The dog hit on active. Operator ended test. Dog demonstrated a positive hit on the active thermos.
VII. Conclusion Six out of seven of the trained odor detection dogs were able to clearly identify and hit on SEQ ID NO: l with the same action for which they would make for a Parkinson's disease positive t-shirt. In these 6 instances, the dogs were acting precisely as if they would be acting if they detected a t-shirt worn by a person with a diagnosis of Parkinson's disease, or suspected Parkinson's disease, and/or Parkinson disease symptoms.
Although the invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.

Claims (43)

Claims What is claimed is:

1. An isolated Parkinson Associated Factor (PAF) protein comprising a polypeptide, wherein the polypeptide comprises an amino acid sequence selected from.:
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

2. A nucleic acid encoding a polypeptide of claim 1.

3. An isolated protein comprising a polypeptide, wherein the polypeptide consists of an amino acid sequence selected from: SEQ ID NO: 1, SEQ 11D NO: 2, SEQ ID
NO: 3 and SEQ ID NO: 4.

4. A protein of claim 3, wherein amino acid residue 12 is geranylgeranylcysteine with an attached carboxymethyl group or farnesylcysteine with an attached carboxymethyl group.

5. A protein of claim 3, wherein amino acid residue 12 is farnesylcysteine with an attached carboxymethyl group.

6. A method of assessing a Parkinson's disease status in an individual, comprising the steps of obtaining a biological sample from the individual; obtaining a biomarker level for a biomarker comprising Parkinson Associated Factor (PAF) protein to comprise biomarker information from the individual; comparing the biomarker information from the individual to a reference biomarker information set corresponding to a known Parkinson's disease status; and categorizing the individual as having the Parkinson's disease status if the reference biomarker information of the individual does not differ significantly from the reference biomarker information set.

7. The method of claim 6, wherein obtaining a biological sample comprises drawing gaseous constituents of a closed space above skin of the individual.

8. The method of claim 6, wherein the biomarker information comprises age information for the individual.

9. The method of claim 6, wherein the Parkinson Associated Factor (PAF) protein comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 wherein amino acid residue 12 is a prenylated cysteine with an attached carboxymethyl group.

10. The prenylated cysteine of claim 9, wherein the cysteine is a geranylgeranylcysteine.

11. The prenylated cysteine of claim 9, wherein the cysteine is a farnesylcysteine.

12. The method of claim 6, wherein the categorizing has a medically actionable sensitivity and specificity.

13. The method of claim 6, comprising transmitting a report of results of the categorizing to a healthcare professional.

14. The method of claim 6, wherein the report recommends that a Parkinson's disease diagnostic clinical evaluation be performed.

15. The method of claim 6, wherein the individual undergoes a Parkinson's disease diagnostic clinical evaluation.

16. The method of claim 6, wherein the obtaining protein levels comprises contacting a fraction of circulating blood or cerebrospinal fluid sample to a set of antibodies, wherein the set of antibodies comprises antibodies specific to a Parkinson Associated Factor (PAF) protein.

17. A method of monitoring efficacy of a Parkinson's disease treatment regimen in an individual, comprising the steps of obtaining a first sample comprising gaseous constituents of a closed space above skin from the individual at a first time point;
administering the treatment regimen to the individual; obtaining a second sample comprising gaseous constituents of a closed space above skin from the individual at a second time point; obtaining a first biomarker level comprising protein levels for one or more Parkinson Associated Factor (PAF) proteins in the first sample and a second biomarker level comprising protein levels for one or more Parkinson Associated Factor (PAF) proteins in the second sample, to comprise biomarker information for the first sample and the second sample; wherein a change in protein levels indicates efficacy of the Parkinson's disease treatment.

18. The method of claim 17, wherein obtaining the first sample comprises drawing gaseous constituents of a closed space above skin of the individual.

19. The method of claim 17, wherein the treatment regimen comprises an anti-Parkinson agent.

20. The method of claim 17, wherein the Parkinson Associated Factor (PAF) protein comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 wherein amino acid residue 12 is famesylcysteine with an attached carboxymethyl group.

21. The method of claim 17, comprising changing the treatment regimen if no efficacy is indicated.

22. The method of claim 17, comprising repeating the treatment regimen if no efficacy is indicated.

23. The method of claim 17, comprising discontinuing the treatment regimen if efficacy is indicated.

24. A method of assessing a Parkinson's disease risk status in an individual, comprising the steps of obtaining a biological sample from the individual;
obtaining protein levels for a Parkinson Associated Factor (PAF) protein relevant to Parkinson's disease in the sample to comprise biomarker information from the individual;
comparing the biomarker information from the individual to a reference biomarker information set corresponding to a known Parkinson's disease status; and categorizing the individual as having the Parkinson's disease risk status if the reference biomarker information of the individual does not differ significantly from the reference biomarker information set.

25. The method of claim 24, wherein obtaining a biological sample comprises drawing gaseous constituents of a closed space above skin of the individual.

26. The method of claim 24, wherein the information comprises age information for the individual.

27. The method of claim 24, comprising transmitting a report of results of the categorizing to a healthcare professional.

28. The method of claim 24, wherein the individual undergoes a Parkinson's disease diagnostic clinical evaluation.

29. The method of claim 24, wherein the individual undergoes a melanoma assay.

30. The method of claim 24, wherein the obtaining protein levels comprises contacting a fraction of the circulating blood or cerebrospinal fluid sample to a set of antibodies, wherein the set of antibodies comprises antibodies specific to Parkinson Associated Factor (PAF) protein.

31. A method of identifying at least one biomarker PAF protein in a patient for diagnosing a patient with Parkinson's disease or a patient at risk for developing Parkinson's disease comprising the steps of, obtaining a biological sample from the patient; measuring the level of PAF protein in the sample; and optionally comparing the level of PAF protein from the patient to a control sample obtained from an individual whose Parkinson's disease status is known.

32. The method of claim 31, wherein the at least one biomarker PAF protein is selected from SEQ ID NOS: 1-4, optionally wherein the terminal cysteine of any one of SEQ ID NOS: 1-4 is independently a C-terminal geranylgeranylcysteine comprising an attached carboxymethyl group or a C-terminal farnesylcysteine comprising an attached carboxymethyl group.

33. The method of claim 31, wherein measuring the level of PAF protein comprises determining the amount of biomarker PAF protein in the biological sample using mass spectrometry (MS) or immunoassay analysis or both.

34. The method of claim 33, wherein the MS analysis comprises matrix assister laser desorption/ionization (MALDI) time of flight (TOF) MS analysis or electrospray ionization (ESI) MS, tandem mass spectrometry (MS/MS), gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS).

35. The method of claim 33, wherein the immunoassay analysis comprises an enzyme-linked immunosorbent assay (ELISA).

36. A method of detecting the presence or absence of at least one biomarker PAF
protein selected from PAF-1, PAF-2, PAF-3 or PAF-4 in a patient comprising, assaying a biological sample obtained from the patient for the presence or absence of PAF-1, PAF-2, PAF-3 or PAF-4.

37. The method of claim 36, wherein assaying the biological sample comprises contacting the biological sample with antibodies specific for PAF-1, PAF-2, PAF-3 or PAF-4, and detecting the presence or absence of antibodies bound to PAF-1, PAF-2, PAF-3 or PAF-4.

38. The method of claim 36, wherein assaying the biological sample comprises using mass spectroscopy to detect the presence or absence of PAF-1, PAF-2, PAF-3 or PAF-4 in the biological sample.

39. The method of claim 36, wherein assaying the biological sample comprises using gas chromatography to detect the presence or absence of PAF-1, PAF-2, PAF-3 or PAF-4 in the biological sample.

40. A method of training dogs in odor detection of Parkinson's disease, comprising training a dog to detect a farnesylated synthetic peptide corresponding to a Malassezia species A factor when present on a surface.

41. The method of claim 40, wherein the farnesylated synthetic peptide is SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

42. The method of claim 41, wherein the farnesylated synthetic peptide is SEQ ID
NO: 1.

43. A method of claim 41, wherein the surface is an article of clothing.