摘要:一位2歲男孩從3個月大開始有反覆感染, 發燒, 皮膚紅疹, 血小板低下, 小腸發炎合併生長遲緩, 中樞系統發炎, 抽筋, 發展遲緩, 反覆性軟骨炎與附睪炎. 我們利用全表現子序列分析顯示他帶有c.334_335delTA, p.I112Wfs*3 heterozygous proteasome maturation protein (POMP) 蛋白酶體成熟蛋白基因突變, 且為全新突變. 蛋白酶體成熟蛋白的功能為蛋白酶體proteasome的攜帶蛋白, 促進蛋白酶體成熟. 實驗顯示抑制POMP蛋白會讓蛋白酶體組合不良, 蛋白酶體功能下降, 抗原呈現經由第一型MHC分子降低, 蛋白酶體次單元缺損之小鼠實驗顯示胸線發育缺損, T細胞發育受影響, CD8+ T細胞減少. 目前沒有POMP缺損小鼠之研究數據可以佐證蛋白酶體缺損將造成免疫缺損或自體發炎, 因此我們提出假說 (1) 蛋白酶體成熟蛋白POMP homozygous 基因突變可能為胚胎死亡, 而heterozygous POMP突變得以存活; (2)POMP基因突變將導致POMP蛋白結構受損, 蛋白酶體功能下降; (3) POMP缺損影響胸腺發育, 抗原呈現, 與CD8+ T細胞成熟和存活; (4)蛋白酶體缺損將導致過多發炎反應, 透過增強的干擾素系統或NF-B訊息傳遞異常. 導致自體發炎. 我們將利用CRISPR/Cas9基因編輯技術, 產生帶有 heterozygous c.333_334delCA, p.I112Wfs*3 POMP基因突變之小鼠, 我們設計與標的基因20個核苷酸互補之guide RNA, Cas9 mRNA, 和帶有POMP I112Wfs*3之DNA模板基因非同源互換, 經由顯微注射入小鼠胚胎, 再挑選出帶有POMP基因突變的小鼠, 藉由此POMP缺損小鼠的疾病模式, 我們可以探討致病原理, 免疫缺損或發炎的機轉, 我們並利用POMP缺損小鼠發展評估JAK1/2抑制劑的治療效果, JAK1/2抑制劑抑制細胞激素的反應, 並已應用於發炎疾病, 血液疾病與癌症, 但對於POMP缺乏症之療效尚不清楚, 本研究最終目的是希望能將研究結果應用於病人治療.
Abstract: A two-year-old male patient had recurrent infections, fever, skin rashes, thrombocytopenia, intestinal inflammation with failure to thrive, central nervous system vasculitis, seizure, developmental delay, recurrent chondritis and epididymitis since 3 months old. A de novo c.334_335delTA, p.I112Wfs*3 heterozygous proteasome maturation protein (POMP) gene mutation was found by whole exome sequencing analysis in the patient in a previous study. The function of POMP is a proteasome chaperon and supports proteasome maturation. Knockdown POMP in cell lines showed the impairment of proteasome assembly, reduced MHC class I surface expression and antigen presentation, decreased proteasomal activities and induction of apoptosis. Proteasome subunit proteins genes knock-out mice showed defects in positive and negative selection in the thymus and a profound defect in the generation of CD8+ T cells. There are no POMP deficient mice model to demonstrate the immunodeficiency and autoinflammation due to POMP gene mutation and proteasome defect. We hypothesized that (1) POMP heterozygous mutation is sufficient for survival (total 3 patients identified in the world) and homozygous mutation may be embryonal lethal; (2) POMP mutation will produce a truncated POMP protein and decrease proteasome function; (3) defects the immunoproteasome and thymoproteasome will affect positive and negative selection in thymus, MHC class I restricted antigen presentation, CD8+ T cells differentiation and survival; (4) proteasome defects will lead to decrease NF-kB signaling and/or enhance interferon (IFN) associated auto-inflammation. To address our hypothesis, we plan to generate a mice model carrying heterozygous c.333_334delCA, p.I112Wfs*3 POMP mutation, the same as c.334_335delTA, p.I112Wfs*3 POMP mutation in our patient, using CRISPR/cas9 gene editing technology. Gene editing based on CRISPR/Cas9 can be directed by guide RNA (gRNA) containing a 20-nt targeting sequence complimentary to the target gene sequence to induce cleavage and gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR). We plan to design and package the guide RNAs, donor template carrying the POMP gene p.I112Wfs*3 mutation and Cas9 mRNA into a plasmid. After screening the gRNAs gene editing effect in the cell lines, we will generate the heterozygous transgenic mice by microinjection of gRNAs, donor templates and Cas9 mRNA to the mice embryo. Based on the POMP deficient transgenic mice model, we can investigate the disease pathogenesis, mechanism of immune deficiency and anto-inflammation. We also plan to evaluate the therapeutic effect of cytokine inhibition using JAK1/2 inhibitor for the autoinflammation from POMP deficiency. JAK1/2 inhibitors suppress the signaling of many cytokines, notable IFN-gamma, IL-2 and IL-6. JAK1/2 inhibitor (ruxolitinib) has been approved effective for the treatment a variety of inflammatory or hematology diseases and cancer. The therapeutic effect is unclear for POMP deficiency. Ultimately, our research findings will benefit our patients and family.