Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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
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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
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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.
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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
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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
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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
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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,
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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.
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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.
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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
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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
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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-
2
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
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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
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(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
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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
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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.
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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
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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
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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.
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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
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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.
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= 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
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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.
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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.
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