Precision Medicine for Sng/Pain Control

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). The musculoskeletal disorders are not only the most common cause of chronic sore/pain, but they also result in significant disability in about 50% of sufferers in the US. Furthermore, since these disorders are the most common cause of severe, long-term pain and disability in the elderly over 65 years. In the practice of pain medicine, the doctors predominantly use multi-modal treatment, including medications, physical agents, and injection, to relieve patients' discomfort.

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 sore/pain in fibromyalgia.

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 investigators can determine the optimal treatment modality by genetic biomarkers, the treatment course and total expanse will decrease a lot.

Investigators 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) could be the prognostic biomarkers of sore /pain treatments. Our specific Aims are:

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

Six. study design I Patient eligibility Investigators will recruit patients from National Taiwan University Hospital Bei-Hu Branch.

Inclusion criteria

(1) Age between 20-100 years old. (2) 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.

Exclusion criteria

Those having active infection, malignancy, and hematological diseases were excluded. The patients had received local injection at upper trapezius within 3 months are also excluded.

II Study design and flow

• After obtaining the informed consent, the basic demographic data, including age, gender, job, education level, and past medical history of eligible patients are collected.
• The eligible patients first received LLLT with a 685-nm wavelength and an output of 30 mW 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.
• Then, they are randomly 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.
• The recruited patients receive evaluation before and after injection, and 2-week after injection. The primary outcome is VAS-pain and VAS-sore (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. 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.
• Rescue therapy (cross-over treatment): If the participant does not satisfy with their first round treatment and the improvement of VAS is less than 1.0, 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.

(1) 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, and subjected to agarose gel and O.D. ratio tests to confirm its purity and concentration. DNA will be fragmented using Covaris, aiming at the peak length of 800 bp. Illumina libraries will be generated from gDNA using TruSeq Library Preparation Kit.

DNA capture probes will be custom-designed to target 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 and will be synthesized using the Roche KAPA HyperChoice protocol. 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.

(2) Metabolomic and proteomic analysis

• Briefly, The LC-MS analyses of urine and serum involved using an Agilent 1290 UPLC system (ACQUITY UPLC HSS T3 column, 2.1×100 mm; 1.8 µm; Waters, Milford, MA, USA) coupled with the 6540-Quadrupole-Time-of-Flight (QTOF) mass system (Agilent Technologies, Santa Clara, CA, USA). MS raw files were converted to the mzXML format using Trapper (ISB) and processed by TIPick, an in-house package. After TIPick processing, the scaling-based normalization was performed according to the total ion abundances from each UHPLC-MS data set.
• The LC-MS lipidomic profiling was performed and described in detail. Concisely, lipidomic profiling was run on a ZORBAX Eclipse Plus C18 (2.1 x 100 mm, 1.8 µm, Agilent Technologies, Waldbronn, Germany) for QTOF, as well as mobile phase A consisted of 0.1% aqueous formic acid and 10 mM ammonium acetate and mobile phase B consisted of 0.1% formic acid and 10 mM ammonium acetate in ACN/isopropyl alcohol (50/50). The autosampler and column oven were maintained at 4°C and 55°C, respectively. The injection volume was 5 μl. MS acquisition was executed in the precursor ion scan (PIS) mode and multiple reaction monitoring (MRM) mode.
• All UPLC-MS raw data were converted to mzXML format by using Trapper (ISB) and normalized by TIPick, an in-house package, as well as peak enhancement and peak chosen for the targeted metabolites. An in-house database of sphingomyelins (SM), lysophosphatidylcholine (LysoPC), ceramides (Cer), phosphatidylcholines (PCs), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and cerebroside (CB) in the Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, was used for screening.

Inclusion criteria

• (1) Age between 20-100 years old.
• (2) Diagnosed as myofascial pain syndrome patients and willing to receive treatment (including LLLT, therapeutic ultrasound, and local dextrose injection therapy).

Exclusion criteria

• Those having active infection, malignancy, and hematological diseases were excluded. The patients had received local injection at upper trapezius within 3 months are also excluded.