Patent Application
20220042099
Field of the Invention Early diagnosis, prognosis, and monitoring of Parkinson's disease (“PD”). Treatment regimens based on these diagnostic methods and on modulation of miRNA associated with Parkinson's disease.
Description of Related Art Parkinson's disease is a long-term degenerative disorder of the central nervous system that mainly affects the motor system.[1] The symptoms generally come on slowly over time.[1] Early in the disease, the prominent symptoms are shaking, rigidity, slowness of movement, and difficulty with walking.[1] Thinking and behavioral problems may also occur and dementia becomes common in the advanced stages of the disease.[2] Depression and anxiety are also common occurring in more than a third of people with PD.[2] Other symptoms include sensory, sleep, and emotional problems.[1][2] The main motor symptoms are collectively called “parkinsonism”, or a “parkinsonian syndrome”.[4] The cause of Parkinson's disease is generally unknown, but believed to involve both genetic and environmental factors.[4]
Parkinson's disease typically occurs in people over the age of 60, of which about one percent are affected.[1][3] Males are more often affected than females.[4] When it is seen in people before the age of 50, it is called young-onset PD. The average life expectancy following diagnosis is between 7 and 14 years.[2]
The cause of Parkinson's disease is generally unknown, but believed to involve both genetic and environmental factors.[4] Those with a family member affected are more likely to get the disease themselves.[4] There is also an increased risk in people exposed to certain pesticides and among those who have had prior head injuries, while there is a reduced risk in tobacco smokers and those who drink coffee or tea.[4] The motor symptoms of the disease result from the death of cells in the substantia nigra, a region of the midbrain resulting in an insufficient dopamine level[1]. The reason for this cell death is poorly understood, but corresponds to the build-up of proteins into Lewy bodies in the neurons.[4] Diagnosis of typical cases is mainly based on symptoms, with tests such as neuroimaging being used to rule out other diseases.[1]
There is no cure for Parkinson's disease so treatment is directed at improving symptoms. Initial treatment is typically with the antiparkinson medication levodopa (L-DOPA), with dopamine agonists being used once levodopa becomes less effective.[2] As the disease progresses and neurons continue to be lost, these medications become less effective while at the same time they produce a complication marked by involuntary writhing movements.[2] Diet and some forms of rehabilitation have shown some effectiveness at improving symptoms. Surgery to place microelectrodes for deep brain stimulation has been used to reduce motor symptoms in severe cases where drugs are ineffective.[1] Evidence for treatments for the non-movement-related symptoms of PD, such as sleep disturbances and emotional problems, is less strong.[4]
Early diagnosis of Parkinson's disease as well as factors contributing to its development and progression would be desirable because it would permit earlier and targeted therapeutic intervention. However, discovery of reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue such as the inability or risk to biopsy or test tissue from the central nervous system directly. Prior attempts have been made to profile mi-RNA (micro-RNA) in serum or cerebrospinal fluid (“CSF”) to associate particular markers with PD[5]. And prior work has looked at a possible role of a brain-gut-microbiota axis dysregulation in Parkinson's disease[6]. Another study focused on the identification of a protein marker (salivary protein DJ-1) as a potential PD biomarker[7]. In view of the need for reliable, sensitive, specific, comparable, and accessible markers, or panels of markers, that correlate with PD as well as its etiology and/or symptoms, the inventors investigated correlations between miRNA and microbial RNAs in saliva.
A method for detecting, diagnosing, prognosing, or monitoring Parkinson's Disease comprising detecting abnormal levels of one or more miRNA and/or microbial RNAs in the saliva of a subject, and optionally, when an abnormal level is detected, further evaluating or testing the patient for Parkinson's Disease or treating the subject for Parkinson's Disease.
Given the complexity of PD, the difficulty in diagnosis and the inaccessibility of the nervous tissue, particularly the brain, to repeated sampling; the development of accessible biomarkers which can serve as indicators or sensors of the underlying pathophysiological processes is needed. From the extensive motor, cognitive, psychiatric, and autonomic symptoms, it is clear that multiple brain regions and peripheral tissues are affected in PD. Thus, the possibility that certain alterations in protein biomarkers can be used to diagnose PD was investigated.
The identification of biochemical markers that are easily accessible and accurately measured would represent a great advance in the diagnosis and treatment of PD. Since biological fluids such as blood and saliva are the most accessible and routine physical source of biological material available for diagnostic testing, saliva was mined for biomarkers for PD diagnostic development and testing.
Embodiments of the invention assessed the prevelance of numerous markers found in saliva as biomarkers for PD.
Saliva is a slightly alkaline secretion of water, mucin, protein, salts, and often a starch-splitting enzyme (as ptyalin) that is secreted into the mouth by salivary glands, lubricates ingested food, and often begins the breakdown of starches. Saliva is released by the submandibular gland, parotid gland, and/or sublingual glands and saliva release may be stimulated by the sympathetic and/or parasympathetic nervous system activity. Saliva released primarily by sympathetic or parasympathetic induction may be used to isolate microRNAs.
Saliva may be collected by expectoration, swabbing the mouth, passive drool, or by other methods known in the art. In some embodiments it may be withdrawn from a salivary gland. In some embodiments, a saliva sample may be further purified, for example, by centrifugation or filtration. For example, it may be filtered through a 0.22 micron or 0.45 micron membrane, and all membrane sizes in between, and the separated components used to recover microRNAs. In other embodiments, proteins or enzymes that degrade microRNA may be removed, inactivated or neutralized in a saliva sample.
Some representative, but not limiting saliva collection and miRNA purification procedures include purifying salivary RNA in accordance with, for example, the Oragene RNA purification protocol using TRI Reagent LS, a TriZol purification method, or similar method. The Oragene purification protocol generally includes multiple parts. In the first part, a sample is shaken vigorously for 8 seconds or longer and the sample is incubated in the original vial at 50° C. for one hour in a water bath or for two hours in an air incubator. In the second part, a 250-500 μL aliquot of saliva is transferred to a microcentrifuge tube, the microcentrifuge tube is incubated at 90° C. for 15 minutes and cooled to room temperature, the microcentrifuge tube is incubated on ice for 10 minutes, the saliva sample is centrifuged at maximum speed (>13,000×g) for 3 minutes, the clear supernatant is transferred into a fresh microcentrifuge tube and the precipitate is discarded, two volumes of cold 95% EtOH is added to the clear supernatant and mixed, the supernatant mixture is incubated at −20° C. for 30 minutes, the microcentrifuge tube is centrifuged at maximum speed, the precipitate is collected while the supernatant is discarded, the precipitate is dissolved in 350 μL of buffer RLT, and 350 μL of 70% EtOH is added to the dissolved pellet mixture and mixed by vortexing. The first two parts may be followed by the Qiagen RNeasy cleanup procedure.
The purification process may further include a second purification step of, for example, purifying the saliva sample using a RNeasy mini spin column by Qiagen. The purification of a biological sample may include any suitable number of steps in any suitable order. Purification processes may also differ based on the type of a biological sample collected from the subject. The yield and quality of the purified biological sample may be assessed via a device such as an Agilent Bioanalyzer, for example, to determine if the yield and quality of RNA is above a predetermined threshold.
microRNA or miRNA is a small non-coding RNA molecule containing about 22 nucleotides, which is found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression (see Ambros et al., 2004; Bartel et al., 2004). MicroRNAs affect expression of the majority of human genes, including CLOCK, BMAL1, and other circadian genes. Notably, miRNAs are released by cells that make them and circulate throughout the body in all extracellular fluids where they interact with other tissues and cells. Recent evidence has shown that human miRNAs even interact with the population of bacterial cells that inhabit the lower gastrointestinal tract, termed the gut microbiome. Moreover, circadian changes in the gut microbiome have recently been established. Small non-coding RNAs (miRNAs) suppress protein expression and that have emerged as useful biomarkers in cancer, diabetes, neurodevelopmental, and neurodegenerative disorders. Although miRNAs are made in all tissues and organs of the body, many of them show tissue-specificity. Moreover, miRNAs can act within the cells that synthesize them or be released into the extracellular space (EC) and travel in biofluids to affect other cells. Numerous studies have shown that miRNA expression profiles differ between healthy and diseased states, and that the release of miRNAs into the EC appears elevated following tissue damage. Epigenetic data includes data about miRNAs. Among the objectives of the inventors were to establish the relationship between peripheral measures of miRNA, objective assessment of likely mTBI severity, and sensitive indices of balance and cognitive function.
A miRNA standard nomenclature system uses the prefix “miR” followed by a dash and a number, the latter often indicating order of naming. For example, miR-120 was named and likely discovered prior to miR-241. A capitalized “miR-” refers to the mature form of the miRNA, while the uncapitalized “mir-” refers to the pre-miRNA and the pri-miRNA, and “MIR” refers to the gene that encodes them. The prefix “hsa-” denotes a human miRNA.
The sequences of miRNAs are known and may be obtained by reference to MirBase, Hyper Text Transfer Protocol (HTTP)://WorldWideWeb.mirbase.org/blog/2018/03/mirbase-22-release/ (last accessed Mar. 19, 2018, incorporated by reference) and/or to Hyper Text Transfer Protocol (HTTP)://WorldWideWeb.mirbase.org/index.shtml (last accessed Mar. 19, 2018; incorporated by reference).
miRNA elements. Extracellular transport of miRNA via exosomes and other microvesicles and lipophilic carriers is an established epigenetic mechanism for cells to alter gene expression in nearby and distant cells. The microvesicles and carriers are extruded into the extracellular space, where they can dock and enter cells, and block the translation of mRNA into proteins (Hu et al., 2012). In addition, the microvesicles and carriers are present in various bodily fluids, such as blood and saliva (Gallo et al., 2012), enabling us to measure epigenetic material that may have originated from the central nervous system (CNS) simply by collecting saliva. In fact, the inventors believe that many of the detected miRNAs in saliva are secreted into the oral cavity via sensory nerve afferent terminals and motor nerve efferent terminals that innervate the tongue and salivary glands and thereby provide a relatively direct window to assay miRNAs which might be dysregulated in the CNS of individuals. Thus, extracellular miRNA quantification in saliva provides an attractive and minimally-invasive technique for brain-related biomarker identification in children with a disease or disorder or injury. Moreover, this method minimizes many of the limitations associated with analysis of post-mortem brain tissue or peripheral leukocytes (relevance of expression changes, painful blood draws) employed previously.
miRNA isolation from biological samples such as saliva and their analysis may be performed by methods known in the art, including the methods described by Yoshizawa, et al., Salivary MicroRNAs and Oral Cancer Detection, Methods Mol. Biol., 2013; 936: 313-324 or by using commercially available kits, such as mirVana™ miRNA Isolation Kit).
The microbiome of the gastrointestinal (GI) tract is essential for mammalian physiology, aiding digestion, synthesis, and absorption of important nutritional components such as amino acids, folate, and B vitamins. Accumulating evidence suggests that the GI microbiome also influences host behavior and neurodevelopment through the “microbial-gut-brain axis”. This axis represents an evolving concept of microbial-mediated cross-talk between the central nervous system (CNS) and GI tract that occurs through several different modalities, including direct neural activation, immune modulation, and hormonal, peptidergic, and epigenetic signaling.
microbiome elements. Based on the studies described herein, the inventors have hypothesized that components of the oral microbiome may correlate with the diagnosis of Parkinson's Disease and/or specific behavioral symptoms. As the microbiome can be simultaneously detected using our salivary RNA diagnostic technology, the inventors have evaluated whether inclusion of components of the microbiome would improve diagnostic accuracy for Parkinson's disease. This ability to jointly monitor the miRNA and microbiome elements of the microtranscriptome gains additional significance in view of recent data that miRNA levels can strongly fluctuate in concert with the host microbiome. Moreover, other recent studies have revealed that alterations in the gut microbiome can affect the expression of brain miRNAs in mice, along with the production of anxiety symptoms. Thus, the interaction of the host miRNA elements and the GI microbiome elements and their joint effects on the brain and behavior comprises a key component of our current biomarker discovery path.
Some of the microorganisms as part of the microbiome that can be assessed include at least one of those listed in Table A below:
Lactobacillaceae
Lactobacillus
Lactobacillus acidophilus
Lactobacillus fermentum
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillus salivarius
Lactobacillus mucosae (↑)
L. mucosae
Lactobacillus produces
L. reuteri reduces anxiety
Lactobacilli species
Bifidobacteriaceae
Bifidobacterium
Bifidobacterium animalis
Bifidobacterium dentium
Bifidobacterium longum
Gardnerella
Gardnerella vaginalis
Parascardovia
Bifidobacterium
Parascardovia denticolens
Scardovia
Bifidobacterium produce
B. longum reduces anxious
Bifidobacterium used for
Gardnerella vaginalis is
vaginosis (Chang et al.,
Parascardovia denticolens
Saccharomycetaceae
Candida
Candida alicans
Candida with PD
Candida duliniensis
Sacccaromyces cerevisiae
pora) (↑)
Torulaspora
Candida produces
Torulaspora delbrueckii
Sacccaromyces cerevisiae
Acidaminococcaceae
Acidaminococcus
Acidaminococcus
Acidaminococcus
Vibrionaceae
Brucellaceae
Brucella
Brucella is the cause of
Brucellosis (Alton and
Methylobacteriaceae
Methylobacterium
Nocardiaceae
Rhodococcus
Rhodococcus aurantiacus
Microbacteriaceae
Clavibacter,
Clavibacter michiganensis
Promicromonosporaceae
Cellulosimicrobium
Cellulomicrobium_sp_TH_20
Enterobacteriaceae
Buchnera,
Escherichia produces
Buchnera aphidicola,
Escherichia (↓)
Escherichia is a possible
Enterobacteriaceae is
Rhizobiaceae
Candidatus azobacteroides
Candidatus azobacteroides-
pseidotrichonymphae
Campylobacteraceae
Campylobacter ureolyticus
Campylobacteraceae
Streptococceae
Streptococcus inopinata (↑)
S. mutans contributes to
Streptococcus mutans (↑)
Streptococcus produces
Streptococcus (↑)
Bacillaceae
Baccillus megaterium (↓)
Bacillus produces
Halobacillus mangrove (↓)
Incertae sedis XII (↓)
Incertae sedis XII
Bacillus species reduce
Bacillus sp JPJ produces
Flavobacteriaceae
Capnocytophaga canimorsus
Flavobacteriaceae have
Chryseobacterium (↓)
Wenyingzhuangia (↓)
Wenyingzhuangia fucanilytica
The inventors refined their technique for saliva collection and improved the software/statistical pipeline for RNA processing. As a result, it has become possible to measure both human and non-human RNA within a single sample. This approach has allowed the inventors to define a panel of miRNAs and microbial species that are differentiated in Parkinson's disease, as is described hereinafter. The methods disclosed by the inventors also comprise selecting a set miRNAs, and a set of microbial taxons that can be combined with appropriate weighting coefficients, or used in ratios, to generate a prediction of association with Parkinson's Disease.
Sex and several other biological factors of relevance are considered as potential modifiers of outcome for the utility of our diagnostic tools. Nevertheless, it is absolutely essential that any molecular diagnostic tool that has been developed by the inventors is equally accurate for both males and females. A broad range of clinical, biological and neuropsychological variables are collected at each site on all subjects and specifically examined in all statistical models. Such variables include age, sex, ethnicity, birth age, birth weight, perinatal complications, current weight, body mass index, current oropharyngeal status (allergic rhinitis, sinus infection, cold/flu, fever, dental carries), sleep disorders, gastrointestinal issues, diet, current medications, chronic medical issues, immunization status, medical allergies, dietary restrictions, early intervention services, hearing deficits, visual deficits, surgical history, and family psychiatric history. Rigorous neuropsychological evaluation of the subjects using standardized, age-appropriate and validated measures of Parkinson's indicia may also performed. The results of the inventors” molecular studies are directly compared with all of these in an unbiased manner to determine the specific magnitude of any interacting effects or to test for the presence of associations in the data that might be of interest. The inventors also used a set or a group of patient data to input to the algorithm.
During sleep-wake cycles there are numerous molecular, cellular, and physiological changes that occur. Many of these changes are driven by what are referred to as circadian regulatory genes, such as CLOCK and BMAL1. These, in turn, cause numerous changes in the expression of physiologically relevant genes, proteins, and hormones. Apart from light-dark cycles, the factors that influence expression of circadian genes are not fully understood. Taken together, the inventors' data suggest a previously unknown relationship between saliva miRNA and microbe content as well as temporal influences (i.e., temporal variations) on miRNAs (and/or microbes) themselves. The systems and methods described herein to normalize epigenetic data (sequencing data or other data) that experience temporal variations may be used in any suitable application where temporal variations may affect the data. Thus, in another aspect of the invention, the data set(s) may be normalized to account for temporal variations in sample prevalence. For instance, by determining read-counts of one or more miRNAs or other genetic markers in a biological sample taken from a subject, normalizing epigenetic data of the subject to account for inter-sample read-count variations, wherein the read-count normalization uses one or more invariant miRNAs, determining time of day that the biological sample was taken, and applying an algorithm to the read-count normalized miRNAs, wherein the algorithm uses the time-of-day to normalize the subject's miRNA expression levels relative to time-of-day.
One aspect of the invention is a kit suitable for determining whether a subject has a disease, disorder, or condition including 2 or more miRNA probes of a probe set. Each miRNA probe may include a ribonucleotide sequence corresponding to a specific miRNA described herein. In an implementation, the kit further may include a solid support attached to the 2 or more miRNA probes. In an implementation, the kit may further include at least one of the following: (a) one randomly generated miRNA sequence adapted to be used as a negative control; (b) at least one oligonucleotide sequence derived from a housekeeping gene, used as a standardized control for total RNA degradation; or (c) at least one randomly-generated sequence used as a positive control. Alternatively, a probe set may include miRNA probes having ribonucleotide sequences corresponding to DNA sequences from particular microbiomes described herein.
Another objective of the inventors was to provide a method a method of monitoring progression of a disorder, disease state or injury in a subject, comprising:
analyzing at least two biological samples from the subject taken at different time points to determine a read-count and time-of-day normalized expression levels of one or more specific miRNAs or other genetic markers in each of the at least two biological samples, and comparing the determined levels of the one or more specific miRNAs over time to determine if the subject's read-count and time-of-day normalized expression levels of the one or more specific miRNAs is changing over time, wherein an increase or decrease in the read-count and time-of-day normalized expression levels of the one or more specific miRNAs over time is indicative that the subject's disorder or disease state or injury is improving or deteriorating. In one embodiment, miRNAs subject to time-of-day normalization are selected from the group consisting of Group A circaMiRs and/or those miRNA which share the seed sequences of the Group A circaMiRs.
The analysis can be performed using linear regression analyses, statistical analyses and/or other computer based models of assessing large volumes of data with multiple variables, sometimes each variable being given different weights in the final scoring and/or conclusions based on the data set.
A key aspect of the invention is to identify those subjects at risk for and/or having Parkinson's disease so that a clinician can have more information on a disease that is difficult, at times, to diagnose, particularly in the early stages of the disease. The methodology described herein can be used by itself but preferably with other standard indicia of Parkinson's indicia, for instance, symptoms of Parkinsonism such as bradykinesia, hypokinesia and akinesia.
Thus, once a subject has been determined to be an at risk patient and/or having Parkinson's disease, the subject is treated to reduce and/or attenuate further progression of the disease. Various medications and treatments are known in the art and include Carbidopa-levodopa, Dopamine agonists such pramipexole, ropinirole, rotigotine, and apomorphine, MAO B inhibitors such as selegiline, rasagiline and safinamide, Catechol O-methyltransferase (COMT) inhibitors such as entacapone, anticholinergic medications such as benztropine and trihexyphenidyl, Amantadine, deep brain stimulation, other surgical interventions, prescribed diet and exercise programs
This was a cross-sectional case-control design employing high throughput RNA sequencing to examine salivary microbial RNAs in subjects with early stage Parkinson's disease and healthy age and gender matched controls.
This study was approved by the Institutional Review Board for the Protection of Human Subjects (IRB) at SUNY Upstate Medical University in Syracuse, N.Y. Informed written consent was obtained for all human subjects. Subjects were recruited from the greater Syracuse and Upstate New York area and received copies of the study description, consent documentation, and a comprehensive health and symptom questionnaire packet prior to their study visit. The questionnaire packet encompassed a detailed medical and health history and six standardized instruments: (1) Part I of the Movement Disorder Society—Unified Parkinson's Disease Rating Scale (MDS-UPDRS-I/II) referred to as Non-Motor Aspects of Experiences of Daily Living, (2) Part II of the MDS-UPDRS, referred to as the Motor Experiences of Daily Living, (3) The Scales for Outcomes in Parkinson's Disease Autonomic questionnaire (SCOPA-AUT), (4) The Parkinson's Disease Quality of Life Scale (PDQUALIF), (5) The Non-Motor Symptom Questionnaire (NMS), and (6) The Beck Depression Inventory (BDI).
Subjects included in the Parkinson's disease (PD) group had been previously diagnosed by a neurologist and met the general diagnostic criteria for late-onset PD, including bradykinesia, rigidity, and typically a resting tremor. Exclusion criteria included a history of neuroleptic use or moderate to severe TBI that might have contributed to trauma-induced parkinsonism. Control subjects were included if they had no prior history of major medical procedures or conditions, were never on PD medications or suspected of having a movement disorder, and did not have any first-degree relatives with PD.
All PD subjects were evaluated using Part III of the MDS-UPDRS by a movement disorder specialist or trained Ph.D.-level evaluator. PD subjects also completed a spiral tracing test and cursive handwriting test to screen for persistent non-resting tremor as well as micrographia, and underwent resting tremor measurements in both hands while wearing a highly sensitive accelerometer (sampling frequency=250 Hz). Height, weight, blood pressure and pulse were obtained on all subjects. All subjects then completed a detailed sensory, motor, cognitive, and balance assessment that included: (1) 12-item Modified Brief Smell Identification Test (mBSIT); (2) 10-item taste test (for sweet, salty, sour and bitter solutions); (3) Trailmaking A test; (4) Trailmaking B test; (5) Digit Span Forward test; (6) Digit Span Reverse test; (7) Simple Reaction Time (SRT); (8) Procedural Reaction Time test (PRT); (9) Go/No-Go test (GNG); and (10) balance/body sway measurements (30 seconds duration) with their shoes off in 10 different postures, while wearing an accelerometer around their waste. With the exception of the two sensory measures, these items were part of ClearEdge®, an integrated FDA-listed tablet-based functional assessment system (Quadrant Biosciences, Inc.) that incorporates three simple and complex reaction time measures (SRT, PRT, GNG) from DANA BrainVitals® battery (AnthoTronix, Inc.) along with the measurements of postural sway and cognitive performance. The postures that were used were as follows: Two legs side by side, eyes open, on a hard surface (TLEO); Two legs side by side, eyes closed, on a hard surface (TLEC); Tandem stance, eyes open, on a hard surface (TSEO); Tandem stance, eyes closed, on a hard surface (TSEC); Two legs side by side, eyes open, on a foam pad (TLEOFP); Two legs side by side, eyes closed, on a foam pad (TLECFP); Tandem stance, eyes open, on a foam pad (TSEOFP); Tandem stance, eyes closed, on a foam pad (TSECFP); a simple dual task involving tandem stance, eyes open, on a hard surface while holding the tablet device (TSEOHT); and a complex dual task involving completion of Trailmaking B while holding the tablet, with two legs side by side, eyes open, on a hard surface (TLEOCT).
Raw demographic data were compiled for all subjects. The functional Balance, Motor, and Cognitive score data were converted to z scores by direct comparison of each subject to a trimmed set of data that represented the mean of the control group after removal of any outlier data points (exceeding+/−2 standard deviations) from any of the measures in the control data set. The outlier points were retained however, for the between group comparisons. The resulting set of 35 demographic and functional variables were then screened for normality in PD and controls separately using the Shapiro-Wilk Test. This indicated that more than half of the variables in both subject groups failed the normality test. Accordingly, we used a Mann-Whitney test, with false discovery rate set at FDR<0.1 to identify rank-based differences between PD and control groups. For simplicity, however, all demographic and functional differences reported are either mean percentage or z score differences. The additional clinical and functional data obtained on the PD subjects was compiled as relative frequencies or raw values and instrument scores. These values were cross-referenced where appropriate to established cutoff values for mild to moderate PD symptom severity based on published literature.
Subjects provided a saliva sample by expectoration into an OraGene RNA (RE-100) collection vial (DNA Genotek, Ottawa, ON). At least 30 minutes had elapsed between the time of last food or drink consumption and saliva collection. Before collecting saliva samples, each subject rinsed their mouth with bottled water. Approximately 1 mL of saliva was obtained from each participant. Samples were stored at room temperature during the study visit and then at 4 C until processing. A Trizol method was used to purify the salivary RNA and a second round of purification was followed using an RNEasy mini column (Qiagen). Yield and quality of the RNA samples was assessed with the RNA NanoChip on the Agilent Bioanalyzer prior to library construction using the Illumina TruSeq Small RNA Sample Prep protocol (Illumina; San Diego, Calif.). Identification and quantification of microbial RNA was performed using next generation sequencing (NGS) on a NextSeq 500 instrument (Illumina). Sets of 48 samples were indexed together at a targeted depth of 10 million single-end 75 bp reads per sample. De-indexing, adapter trimming and quality control metrics were obtained from Partek Flow software. Alignment of microbial transcripts was performed using the K-Slam software, which references the NCBI Taxonomy database, after filtering to remove miRNAs and other RNAs that aligned to the human transcriptome. Taxons were defined by their family, genus, species, and subspecies (when available).
The microbial RNA present in raw counts of 10 or more in at least 10% of samples were interrogated for differences between subject groups in overall richness using the Shannon alpha diversity and Bray-Curtiss beta diversity metrics. The set of genus and species data were then examined for between group differences using the Mann-Whitney test with false discovery correction (FDR<0.05) and for the ability to completely distinguish the subjects in a binomial classification test using logistic regression with receiver cperating characteristic (ROC) curve analysis (with 10-fold cross-validation). The biological significance of differential microbial transcript abundance was assessed using KEGG Pathway mapping as well as hierarchical clustering analysis within MicrobiomeAnalyst and MetaboAnalyst R packages. Correlations between different microbial and functional and demographic measures were assessed in an exploratory manner by Pearson product-moment correlation analysis.
A total of 84 subjects completed the study, including 36 healthy controls with no history of movement disorder and 48 subjects with early stage PD (Table 1). None of the participants had active dental caries or periodontal disease.
Among the PD subjects, the average duration of a diagnosis was 3.4 years (SE±0.56 years), with an average Hoehn & Yahr Stage of 1.92, and average scores for subscales of the MDS-UPDRS, NMS, SCOPA-AUT, PDQUALIF, and BDI all falling in established ‘mild’ ranges for those instruments according to published criteria (UPDRS-I 10.0, UPDRS-II 8.6, UPDRS-III 23.9) Most PD subjects (69%) were observed to have resting tremor and 87% were on PD medication (Table 2). Notably, more than 95% of our PD subjects had evidence of upper or lower GI disturbance (Table 2).
Compared with healthy control subjects, the PD subjects in our cohort were found to exhibit significant changes in several indices of motor, cognitive and sensory function. Specifically, the early stage PD subjects showed a significant increase in completion time for the Trailmaking A and B tasks, and a decrease in Trailmaking B completion score (Table 3). These deficits were present in the absence of a significant change in Simple Reaction Time Score, although a trend for slower reaction times was apparent. Complementing these findings, the Procedural Reaction Time (PRT) Score was also significantly decreased in the DANA Brain Vitals set of measures (SRT, PRT, GNG) (Table 3).
Balance Scores. PD subjects were found to exhibit increased body sway (decreased score) in four of the balance measures (TLEO, TLEC, TSEO, TLEOFP) and an apparent increase in the performance of one balance measure (TSECFP) (Table 3). However, inspection of the data for this latter task, which is usually considered the most difficult, indicated that the higher scores were likely due to selection bias of more capable PD subjects, because multiple PD subjects (n=7) were actually unable to complete it. This was also true for another task that is considered nearly as difficult (TSEOFP), where 8 PD subjects were unable to complete it and there was no overall between group difference. Thus, discounting the TSECFP and TSEOFP tasks, the overall trend when the full group scores were available was for reduced balance scores in the PD group.
Reaction Times and Cognitive Scores. PD subjects demonstrated a consistent decrease in their simple and complex reaction time measures compared with Controls, as reflected in reduced performance on the SRT, PRT and GNG tasks (Table 3). Consistent with the slowed reactions times, we also observed that PD subjects took longer to complete the Trailmaking A and B tasks, and this was accompanied by reduced scores on these measures as well (Table 3). For Trailmaking B, this was also true when subjects had to perform the test in a dual task condition, while maintaining upright standing posture (Table 3).
Chemosensory Scores. PD subjects showed highly significant decreased performance in measures of both taste and smell (Table 3). Notably, performance on these two sensory measures was also significantly correlated (Pearson's R=0.27; p=0.015), although this should not be taken as evidence of a strong association between the two measures.
Alpha and Beta Diversity. After filtering to remove taxa that were less consistently observed in the saliva samples, we did not observe any significant differences in overall alpha and beta diversity (
Genus and Species Differences. A total of 50 microbiome taxa exhibited significant differences in abundance in PD subjects compared with control subjects. These included 16 genera and 34 species, and encompassed bacteria, phage, and Eukaryotic taxa (Table 4) (FDR <0.05). The majority of changes observed were increases in abundance (n=36) rather than decreases in abundance (n=14) (Table 4). Notably, 12 of the genera findings had one or more subordinate species findings, while 4 were changed in isolation. Included among the more commonly changed bacteria species were multiple members of the Lactobacillus (n=6) and Bifidobacterium (n=3) genera (Table 4). We also observed a significant decrease in a bacteriophage (Streptococcus phage Phi Spn 200), and significant increases in three yeast species (Candida albicans, Candida dubliniensis, and Saccharomyces cerevisiae) in PD subjects (Table 4).
LACTOBACILLUS
METHYLOBACTERIUM
PARASCARDOVIA
RHODOCOCCUS
SCARDOVIA
TORULASPORA
WENYINGZHUANGIA
To further probe the consistency of the group microbiome differences, we subjected the genus and species level data to logistic regression classification and area under the receiver operating characteristic curve (ROC) analysis. This indicated a strong separation of the groups was possible using a set of the microbiota data transformed into 5 ratios of two taxa plus 4 additional individual taxa (n=11 taxa total), with an area under the curve (AUC) during training of 0.95 and an AUC during 10-fold cross-validation of 0.90 (
In addition to probing for individual transcript alterations, we also tested whether there was evidence of alterations in the expression of microbial metabolic pathways. A total of 167 KEGG pathways were examined, of which 6 showed nominally significant changes (Table 5). The changes included three pathways with increased RNA transcript expression and three with decreased expression.
To judge the consistency of microbial metabolic changes in PD subjects, we used hierarchical clustering of the data within the six altered functional pathways and added whisker-box plots (
Our final analysis probed for significant associations among the microbial data and the full set of 43 medical, demographic, and functional outcome measures in an exploratory fashion using Pearson correlation analysis. Because of the large number of correlations generated, we used a conservative approach in interpreting the results of these exploratory analyses. Here, we focus only on the 10 most robust correlations in magnitude based on absolute rho value (Table 6). Notably, the magnitude of these correlations all exceeded |0.576| with 9 positive correlations (all at the species level) and a single negative correlation (at the genus level). Interestingly, three of the four most robust correlations were with the Duration of PD (years with diagnosis), while the sole negative correlation was with a balance measure (TSEOFP). As already noted, however, this specific functional task did not show significant differences in performance between PD and control groups (Table 3). Among the 10 most robust overall correlations, only one involved a correlation between a significantly changed taxon (Lactobacillus reuteri) and a significantly changed functional outcome measure (Trailmaking A time to completion).
In another data set, the tope 20 Classifiers in Host and Tax Mixed RNA are shown in Table 7.
Vibrio fischeri MJ11
Rhodococcus sp. 008
Paenibacillaceae
Clostridium botulinum A3 str. Loc Maree
The present study defines differences in the oral microbiome in early stage PD as determined from shotgun RNA sequencing of saliva samples combined with detailed phenotypic characterization of subjects. We have six principal findings. First, even in early stage PD, with most subjects on some form of anti-parkinsonian medication, we found evidence of significant (and often highly robust) decreases in balance, sensory, motor and cognitive function. Second, there was no evidence of overall changes in alpha or beta diversity in early stage PD compared with controls. Third, a distinct set of micobial taxa demonstrated consistent changes in sequence abundance at the genus and species level after appropriate correction for multiple testing. Moreover, approximately half of these observed changes fell into clusters of species within the same genera. Fourth, when considered as potential classifiers in a multivariate logistic regression analysis, as few as 11 taxa were found to be capable of distinguishing early stage PD subjects from controls with a 10-fold cross-validated AUC of 0.90 and overall accuracy of 84.5%. Fifth, metabolic pathway analysis of the microbial transcript abundance revealed changes in a distinct subset of biological networks, several of which were highly-related to each other. And sixth, exploratory analyses indicated the presence of highly significant correlations between specific microbiota and specific subsets of functional measures, including a robust correlation between one of the changed microbiota and one of the changed functional measures. In the space that follows, we briefly discuss the importance of these observations.
Motor impairments are part of the hallmark symptoms of PD, including bradykinesia and rigidity, and represent two of the criteria used in its diagnosis. Thus, the slowing of reaction times and resulting increase in z scores for the speed-based performance elements that we observed are not surprising and are highly-consistent with a vast literature on the topic. Related to this, it is possible that slowing of movements contributed to reduced performance on the specific cognitive outcome measures that we utilized (SRT, PRT, Trailmaking A and B). Notably, however, approximately half the trials on the GNG task actually involve withholding a response, so this task might be expected to be less affected in its overall score than a purely-motor score if the primary issue was motor speed alone. However, our data show highly-similar decreases in both SRT and GNG performance (Table 3), suggesting that the decision-making process does exhibit some impairment as well. The involvement of reduced motor speed in decreased cognitive task performance is further strengthened by examination of the z score magnitudes for the Trailmaking A and B tasks, since the completion times changed more than the scores themselves compared to controls. Again, however, the Trailmaking B showed significant score reductions while the Trailmaking A did not. Thus, although we clearly cannot separate motor and cognitive performance changes in our PD subject cohort, there is a suggestion that the additional cognitive demands of a task result in reduced performance that is added to the effect of bradykinesia.
Another hallmark symptom of PD is postural instability. In the motor examination of the UPDRS, this is usually evaluated through examination of the subjects while standing, walking, turning, and following a pull test. In our cohort of early stage PD subjects, very few individuals exhibited any noticeable impairment in postural stability. Nonetheless, of all the functional measures, the largest z score change in PD subjects was increased body sway compared to controls during a simple static balance task performance (TLEO) (Table 3). This intriguing finding suggests that the computerized functional assessment system we have used to assess PD subjects is highly-sensitive for detecting and quantifying changes in postural sway before they might be obvious or apparent to a trained evaluator.
The final functional domain that we evaluated in our subjects was chemosensory in nature (smell and taste), where our PD cohort scored much worse than the healthy control subjects (Table 3). While not considered pathognomonic, decreased olfaction and taste has been well-documented in PD, including early stages of the disease. Notably, similar decreases in chemosensory function have also been consistently found in subjects with Alzheimer's disease, or a history of mild traumatic brain injury (mTBI). Thus, our findings are consistent with the literature on early stage PD and suggest that these measures may represent useful screening tools, when used in combination with other assessments, for identifying subjects at risk for neurodegenerative disease in general.
Comparison of Microbiome Findings with Prior Studies
Investigations of the GI microbiome has become increasingly prevalent in the past few years, especially in the case of PD which presents with multiple GI symptoms along with motor symptoms and where pathological changes may be occurring well before CNS involvement [see O'Mahony, et al., 2015; Pellegrini et al. 2018]. To date, at least a dozen papers have been published on this topic to probe what might be affected in PD. When the results from these 12 studies are compared, several similar changes can be found, even though they were frequently analyzed at varying levels of classification, and most relied on 16S ribosomal RNA gene sequencing for bacterial identification. Specifically, at the family level of classification, despite differences in tissues and fluids tested, there are overlapping findings from many of the studies, though some bacterial families were less consistent. Eleven of the twelve studies analyzed the microbiomes in fecal stool samples, while one also compared the fecal results to those of sigmoidal colon mucosal biopsies, another compared the fecal results to nasal wash samples, and another investigated potential microbiome changes utilizing oral and nasal swabs (Pereira et al., 2017), although they reported almost no consistent differences in their PD subjects.
Despite the considerable differences in methodology and tissue sources, the results of the present study are highly-consistent with many of those seen in other studies. In fact, half (8/16) of the bacterial families that we found altered were reported to be altered in prior studies (Table 7). In this report, we focus on two of these bacterial families (Bifidobacteriaceae and Lactobacillaceae) which showed similar increases across almost all studies to date and merit further discussion.
Generally regarded as “probiotic” in nature, bacteria within the Bifidobacteriaceae family are proposed to have anti-inflammatory properties and potentially serve beneficial purposes (Mulak and Bonaz., 2015). Thus, it is possible that the changes we and other groups have seen may reflect a compensatory mechanism in the GI tract. However, while Lactobacilli are also generally considered probiotic, some members of the Lactobacillaceae family may exert a disease-worsening effect in PD. Specifically, Lactobacillus reuteri, which we found significantly increased in our PD subjects, was shown in a prior study to increase alpha-synuclein release in the ENS as a result of increasing the firing frequency of mesenteric afferent nerve bundles (by decreasing calcium-dependent potassium channel opening and reducing the slow afterhyperpolarization in these neurons) (Perez-Burgos et al., 2013; Kunze et al., 2009; Paillusson et al., 2012). In this light, it is particularly worthwhile to note that our exploratory correlation analysis identified a robust positive correlation between the abundance of Lactobacillus reuteri and slowing of movement (as reflected in increased performance time on the Trailmaking A test) (Table 6). Other evidence also suggests that Lactobacilli might not be particularly beneficial in PD. Specifically, some members of this bacterial family have been shown to reduce ghrelin secretion, which normally regulates nigrostriatal dopamine and is thought to be neuroprotective, and has been previously reported to be reduced in PD patients (Bayliss et al., 2011; Unger et al., 2011). Thus, based on the available data, the consistent increase in Lactobacillaceae we and others have observed in PD may represent a disadvantageous yet consistent event in the disease. This suggestion lies in stark contrast to much of the current opinion regarding Lactobacilli. Indeed, administration of Lactoballicus reuteri has been shown to reduce anxiety and corticosterone secretion in mice (Bravo et al., 2011), and several other Lactobacillus species have proven beneficial in the treatment of constipation, diarrhea, and IBS symptoms (Fijan, 2014) (see Table 7). Accordingly, we suggest that a closer examination of the benefits and risks of Lactobacillus supplementation is warranted.
Other findings in our PD cohort are worth noting because of their possible relevance to PD and brain function. Among these include changes in several bacterial families that are known to directly affect neurotransmitter levels. These include Lactobacillus and Bifidobacterium genus members already discussed, which produce GABA and acetylcholine (Cryan and Dinan, 2012), Enterobacteriaceae family members, which produce norepinephrine and serotonin and are associated with postural instability and gait difficulty phenotypes in PD (Scheperjans et al., 2015), and members of the Bacillus genus that produce noradrenaline and dopamine (Cryan and Dinan, 2012). Perhaps the most intriguing finding, however, concerns that of the family Nocardiaceae, which includes the Rhodococcus genus and was increased in our early stage PD subjects. The administration of Rhodococcus aurantiacus in laboratory mice was shown to induce encephalitis and cause a movement disorder, due to T-cell mediated inflammation, that subsequently responded in a favorable way to L-DOPA treatment (Min et al., 1999) (Table 7). Thus, the combined set of bacterial families that we observed changed in early stage PD may have broad implications for understanding the pathophysiology of the disorder.
Finally, it is also worthwhile to note that several of the altered microbiota we observed have been linked to PD or are known to play roles in oxidative metabolism. These include members of the Saccharomycetaceae family (encompassing the Candida and Saccaromyces genera), and members of the Acidaminococcaceae and Flavobacteriaceae families (Table 7). Specifically, Candida members produce serotonin and have been anecdotally associated with PD symptoms (Cryan and Dinan, 2012). In contrast, Sacccaromyces cerevisiae produces the rotenone-insensitive NADH:ubiquinone oxidoreductase protein (Ndi1p) which can restore function in complex 1 of the mitochondrial electron transport chain (ETC) that occur due to Pink1 mutations (Vilain et al., 2012). And Acidaminococcus consumes glutamate which is important for oxidation in the intestinal epithelium and is a key contributor to oxidative and amino acid metabolism (Gough et al., 2015). These individual taxon findings are further strengthened by the results of our metabolic pathway findings, which highlighted decreases in Tryptophan and Krebs cycle metabolism and increases in Glycolysis and Pentose phosphate metabolism in early stage PD. Reductions in Tryptophan metabolism could easily lead to reduced serotonin, melatonin and kyenurenate levels, which have all been shown to have neuroprotective properties. And reduced Krebs cycle metabolism could easily lead to overall decreases in ATP production and increased oxidative stress. Viewed this way, the increased Glycolysis and Pentose phosphate pathway activities, could therefore represent compensatory attempts to boost ATP production as well as NADPH levels, with a resulting elevation in reduced glutathione levels leading to greater antioxidant capability. Clearly, further studies are needed to test these suggestions and further characterize the metabolomic profiles of the oral microbiome in early stage PD.
As shown by the results of this study which are disclosed herein, the method according to the invention provides a sensitive, specific, and convenient way to diagnose Parkinson's disease.
All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.
The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references, and does not constitute an admission as to the accuracy of the content of such references.
This application claims the benefit of U.S. provisional application No. 62/747,383 filed Oct. 18, 2018. The contents of U.S. Provisional 62/607,792, filed Dec. 19, 2017 AND U.S. Provisional 62/622,319 filed Jan. 26, 2018 are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/056884 | 10/18/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62747383 | Oct 2018 | US |
