고분해능 질량분석기 기반의 정량적 단백체 기법을 활용한 피질하 경색과 백질뇌증을 동반하는 상염색체 우성 뇌동맥질환 특이적 혈장 바이오마커 발굴 연구

Abstract

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a rare genetic disorder that affects small blood vessels in the brain, leading to various neurological symptoms. The most common symptoms are abnormal cerebral white matter morphology and leukoencephalopathy, and cerebral ischemia, cognitive impairment, emotional lability, migraine, and stroke also occur frequently. The best method for diagnosing CADASIL is through genetic testing for NOTCH3 mutations, but genetic testing may not always be possible or feasible, and not all CADASIL patients have pathogenic NOTCH3 mutations. Therefore, there is a need for research to discover blood-based biomarkers that can aid in the diagnosis and treatment of CADASIL. This study aims to contribute to the improvement of the quality of life for CADASIL patients by identifying biomarkers for diagnosis and monitoring through plasma protein analysis. The study also aims to provide evidence for the management and treatment of CADASIL. Plasma samples were collected from three groups of subjects with similar ages (67 ± 12.5 years) with 5 males and 5 females in each group (CADASIL patient group, stroke patient group, and healthy control group, total of 10 subjects for each group). The collected plasma samples underwent pretreatment, in which the top 14 high-concentration plasma proteins were removed using the Multi Affinity Removal System Human 14 (MARS14) column (100 × 4.6 mm, Agilent Technology, Palo Alto, CA, USA). The remaining plasma proteins that were not removed by the column were harvested and digested using Suspension Trap (S-Trap) digestion method with trypsin/LysC at 37℃ for 16 hours. Using the Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Waltham, MA, USA) and Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific), spectrum was acquired for quantitative analysis of 1,902 plasma proteins using Proteome Discoverer (Thermo Fisher Scientific, ver 2.3) equipped with the Sequest HT algorithm. From this analysis, quantitative and qualitative information for each group was obtained, and differentially expressed proteins (DEPs) were identified. And principal component analysis, Venn diagram analysis, and volcano plot analysis were performed for additional statistical result. Candidate pathways were analyzed through gene ontology (GO) analysis using DEP information. The expression results of potential CADASIL plasma biomarkers identified in this study were compared with those from previous studies. From the result, small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was found to be significantly increased in CADASIL patients compared to both groups, while Treacle protein (TCOF1) showed a decreasing trend. These protein candidates may be utilized for CADASIL screening and monitoring using patient plasma with further validation studies and are expected to contribute to the management and treatment of CADASIL patients.