Functional Genomic Study of Tyrosine Hydroxylase rs6356 for Chronic Pain in Sickle Cell Disease

Patients with sickle cell disease (SCD) suffer from recurrent episodes of acute pain due to vaso-occlusive crisis and persistent chronic pain. The dopaminergic system has not been extensively studied in chronic pain. Tyrosine hydroxylase (TH) is the rate-limiting enzyme of catecholamine biosynthesis. In our ongoing pharmacogenomics study, one single nucleotide polymorphism (SNP) rs6356 (NM_000360.4: c.241C>T) in the tyrosine hydroxylase gene was found to be associated with chronic pain in sickle cell patients. In this study, rs6356 (V81M) variation in tyrosine hydroxylase was found to have an inhibitive effect in its enzymatic activity, and TH-81M was measured with a greater Km value, which represented a minor enzyme-substrate affinity than TH-81V. SH-SY5Y-rs6356 cell line was generated via CRISPR/Cas9 genomic editing in order to evaluate the influence of TH-V81M mutation in subsequent cellular pathways. It was shown that SH-SY5Y-rs6356 cells manifested a lower intensity of dopamine release in response to DMPP-induced acetylcholinergic activation. Further, SH-SY5Y-rs6356 cells also showed correspondingly mitigated activation of cAMP/PKA signaling, which could be exclusively blocked by the D1/D5 antagonist. In addition, V81M tyrosine hydroxylase showed a lower phosphorylation level when PKA was partially activated. We also found some evidence that peripheral PKA inhibition relieved the pain behavior related to thermal and mechanical stimuli in sickle cell mice.