Precision Medicine for Nociception, Sngception and Proprioception.

Description

Precision medicine is defined as "an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person" by the Precision Medicine Initiative.

Pain is defined as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage" by the International Association for the Study of Pain (IASP).Traditionally, soreness, or sng, is also included as one of the pain sensation. Recently, we defined sngception as acid and/or soreness sensation in the somatosensory system, and revealed that analgesia in muscle is through substance P and Tac1.

According to the clinical outcome, some patients responded to physical agents well, and some preferred injections. The genetic variants of the above-mentioned genes might be the determining factors of differential therapeutic effects. However, it took about 4-8 weeks for a patient to switch from one treatment option to another one. If we can determine the optimal treatment modality by genetic biomarkers, the treatment course and total expanse will decrease a lot.

We hypothesize that the genetic variants of the proposed genes (TRPV1, ASIC1a, ASIC3, Tac1, COMT, TCL1A, POMC, RGS4, ASIC2, ASIC4, TRPA1, NK1R, G2A, GPR4, OGR1, TDAG8, TASK1, TASK2, TASK3, TREK1, P2X2, P2X3, P2X5, TRPV4, KCNK1, NTSR1, NTSR2, CaV3.2, Nav1.1, Piezo1, and Piezo2, Runx3 or Egr3, endothelin converting enzyme-like 1 (ECEL1), myosin heavy chain genes MYH3 and MYH8, myosin-binding protein C gene, MYBPC1, and TNNI2, TNNT3 and TPM2 that encode the muscle regulatory proteins troponin I, troponin T and beta-tropomyosin) could be the prognostic biomarkers of sng/pain treatments or proprioceptive function.

1.Specific Aims

1. To set up next generation sequencing (NGS)-based approach to find genetic variants which can determine the response of sng/pain treatment modalities and the phenotype of idiopathic scoliosis.
2. To find possible metabolomics and proteomic markers of sng/pain.
3. To determine the algorithm of precision medicine for sng/pain control via the genetic markers.

2.In the past years, our team had some achievements to justify the search of genetic variants for development of a new sore/pain treatment algorithm.

1. Analgesia of LLLT is through TRPV1.
2. Analgesia by therapeutic ultrasound is through ASIC3.
3. Dextrose injection decreased chronic muscle pain through ASIC1a.
4. Biomarker for sng/pain in fibromyalgia.

3.Study design

(1) Participants from cohort A-Myofascial pain syndrome: We will recruit 80 patients from National Taiwan University Hospital and its Branch hospital.

1. Patient eligibility:
2. Inclusion criteria: (1) Age between 18-100 years old. (2) VAS>=30 or VAS>=30 at 4 kg pressure (3) Diagnosed as myofascial pain syndrome patients and willing to receive treatment (including LLLT, therapeutic ultrasound, and local dextrose injection therapy). The diagnosis of MPS was confirmed by the Principal Investigator using the criteria of taut band, trigger point, and radiating pain.
3. Exclusion criteria: Those having active infection, malignancy, and hematological diseases were excluded. The patients had received local injection at upper trapezius within 6 months are also excluded.
4. Study design:
5. After obtaining the informed consent, the basic demographic data, including age, gender, job, education level, and past medical history of eligible patients are collected.
6. The eligible patients first received LLLT with a 685-nm wavelength and an output of 30 mW (BTL-5000 Laser, BTL, Stevenage, UK) at energy densities of 8 J/cm2 at trigger point of upper trapezius muscle. The pre- and post-treatment VAS-pain and VAS-sng are collected for LLLT phenotype determination, respectively.
7. Then, they are conveniently assigned into two groups (40 subjects in each group): A. therapeutic ultrasound group; B. prolotherapy group. Group A receives 1 MHz therapeutic ultrasound for 5 min at a frequency of 2-3 times per week at the painful upper trapezius muscle. Group B receives hypertonic prolotherapy at perimysium of upper trapezius muscle. The injectant is 5ml 5% dextrose solution. To ensure that the needle was not in a blood vessel, the needle was aspirated before injection. The same physician (the principal investigator) injects all patients to avoid inter-physician variability. No other medication or physical modality was given to avoid efficacy interference in both groups.
8. The recruited patients receive evaluation before and after injection, and 2-week after injection. The primary outcome is VAS-pain and VAS-sng (visual analogue scale) with a score of 0-100, where 100 is the value representing the highest degree of pain. The secondary outcomes are pain threshold, muscle tone, and SF-36-a questionnaire consists of 36 items and 8 domains addressing the patient's perception of their QoL (quality of life). Venous blood and urine samples were collected at first visit and 2-week visit, respectively. The blood samples were labeled with an anonymized ID number, centrifuged, and stored at -80 ºC in a locked freezer until the time of future processing. The buffy coat is separated after centrifugation, and stored as well. The urine samples were aliquoted and stored at -80 ºC in a locked freezer for future analysis.
9. Rescue therapy (cross-over treatment): If the participant does not satisfy with their first round treatment and the improvement of VAS is less than 10, then they are eligible to receive the rescue therapy-the treatment in the other group. And they will return to clinic for another 2 weeks. The outcome variables will be collected in the 3rd visit as well.

(2) Participants from cohort B-idiopathic scoliosis: We will recruit 80 patients from National Taiwan University Hospital and its Branch hospital, and Taichung Veteran Hospital.

1. Patient eligibility:
2. Inclusion criteria: (1) Age between 10-65 years old. (2) Diagnosed as idiopathic scoliosis The diagnosis of scoliosis was confirmed by antero-posterior plain Xray with Cobbs angle larger than 10 degrees.
3. Exclusion criteria: (1) Those having active infection, malignancy, and hematological diseases were excluded. (2) Those having specific etiologies of scoliosis, including congenital scoliosis due to malformation or faulty segmentation of the vertebrae and neuromuscular scoliosis due to muscular imbalance, syndromic scoliosis or degenerative scoliosis.
4. Study design:
5. After obtaining the informed consent, the basic demographic data, including age, gender, job, education level, physical activity level, and past medical history of eligible patients are collected.
6. The recruited patients receive evaluation for once only. The collected parameters include Cobb's angle, spine rotational angle, VAS-pain and VAS-sng (visual analogue scale) with a score of 0-100, where 100 is the value representing the highest degree of pain, pain threshold (Park, Kim et al. 2011), strength and tone of paraspinal muscle, and SF-36-a questionnaire consists of 36 items and 8 domains addressing the patient's perception of their QoL (quality of life)(Li, Wang et al. 2003). Venous blood and urine samples were also collected. The blood samples were labeled with an anonymized ID number, centrifuged, and stored at -80 ºC in a locked freezer until the time of future processing. The buffy coat is separated after centrifugation, and stored as well. The urine samples were aliquoted and stored at -80 ºC in a locked freezer for future analysis.

(3) DNA extraction and NGS-based sequencing and genotyping Genomic DNA will be extracted from peripheral blood mononuclear cells of the participants using the Gentra Puregene kit following the protocol from the manufacturer (Qiagen, Hilden, Mettmann, Germany), and subjected to agarose gel and O.D. ratio tests to confirm its purity and concentration. DNA will be fragmented using Covaris (Covaris, Woburn, MA, USA), aiming at the peak length of 800 bp. Illumina libraries will be generated from gDNA using TruSeq Library Preparation Kit (Illumina, San Diego, CA, USA).

DNA capture probes will be custom-designed to target TRPV1, ASIC1a, ASIC3, Tac1, COMT, TCL1A, POMC, and RGS4, and will be synthesized using the Roche KAPA HyperChoice protocol (Roche, Madison, WI, USA). All the coding regions and non-coding regions (promoters, introns, 5' and 3' untranslated regions) of these 8 genes will be included.

NGS target region enrichment will be applied to enrich/capture the target region (~148 Kb). The enriched libraries will then be sequenced using Illumina MiSeq to generate paired-end reads of 300 bp. The expected depth of the targeted regions will be 200x on average.

Data analyses will be performed as previously described. Briefly, the raw sequencing data will be aligned to the reference human genome (Feb. 2009, GRCh37/hg19) using BWA-MEM. We will use Picard to perform necessary data conversion, sorting and indexing. The main variant calling process, for both single nucleotide variants and indels, will be operated with the GATK software package. Structural variants will also be identified. We will apply ANNOVAR to appropriately annotate the genetic variants; this include at least gene annotation, amino acid change annotation, SIFT scores, PolyPhen2 score, dbSNP identifiers, 1000 Genome Project allele frequencies, gnomAD allele frequencies and ClinVar database with variant clinical significance. We will then use IGV to view the mapping and annotation of sequences on a graphic interface.

The allele frequencies of variants of interest will be compared between different groups of participants. We will look for possible rare variants with very strong effects (the single-gene model) as well as relatively common variants with moderate to strong effects (the complex phenotype model).

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