Determining the pathogenicity of genetic variants in pain-associated genes: Establishment of a refined protocol for patient-derived nociceptors and personalized application in chronic pain patients

对疼痛相关基因的遗传改变筛查越来越多地应用于具有特发性疼痛综合征的患者，但往往导致的结果仍不清楚致病性。鉴于其诊断、治疗和预后的不确定性，这些发现对治疗医生和患者都是巨大的挑战。解开这些变异是否具有临床相关性的唯一可能性是通过研究可能通过诱导多能干细胞（iPSC）产生的患者来源的伤害感受器。然而，这种方法具有神经元分化后几乎不可控的细胞异质性的局限性。这种异质性使得细胞特异性分析的应用变得不可能，也大大降低了所获得数据的意义。在我们的翻译项目中，我们将遇到这两个挑战。通过对基于小纤维病理的慢性疼痛患者进行遗传筛查，我们将确定携带迄今未知致病性遗传变异的患者，并通过iPSC产生个体感觉神经元。结合特定的染色技术，差异FACS分析和电生理表征，我们将分离伤害感受器，这将允许个性化RNA测序，目前受到神经元分化时细胞异质性的阻碍。通过多维度方法和综合数据分析，考虑临床表型和体外数据，我们将识别和表征疼痛相关基因的新型致病变异，并将为治疗医生提供临床使用的表型特征；此外，我们将制定一个完善的方案，以在基础科学中特异性地发现ipsc来源的感觉神经元中的伤害感受器。这个转化项目具有巨大的潜力，通过新的视角和知识丰富和改善临床和基础疼痛研究，对疼痛诊断，治疗和预防产生持续的影响。

Screening for genetic alterations in pain-associated genes is increasingly performed in patients with pain syndromes of so far idiopathic origin and often results in findings that remain of unclear pathogenicity. Given their diagnostic, therapeutic, and prognostic uncertainty, these findings are a great challenge for treating physicians and patients. The only possibility to unravel if such variations are of clinical relevance is by investigating patient-derived nociceptors which may be generated via induced pluripotent stem cells (iPSC). This methodology, however, carries the limitation of almost uncontrollable cellular heterogeneity after neuron differentiation. This heterogeneity renders the application of cell-specific analysis impossible and also dramatically reduces the significance of the obtained data. In our translational project, we will meet these exact two challenges. By genetic screening of a comprehensively characterized patient cohort with chronic pain based on small fiber pathology, we will identify patients carrying genetic variants of so far unknown pathogenicity and will generate individual sensory neurons via iPSC. Combining specific staining techniques, differential FACS analysis, and electrophysiological characterization, we will isolate nociceptors that will allow personalized RNA sequencing which is currently hampered by cellular heterogeneity upon neuronal differentiation. Following a multidimensional approach and by integrative data analysis considering clinical phenotype and in vitro data, we will identify and characterize novel pathogenic variants in pain-associated genes and will provide treating physicians phenotypic features for clinical usage; further, we will formulate a refined protocol to specifically spot nociceptors among iPSC-derived sensory neurons in basic science. This translational project bears the immense potential to enrich and improve clinical and basic pain research by fresh perspectives and knowledge with sustainable impact on pain diagnostics, treatment, and prevention.