| 摘要: | Pearson氏症候群（Pearson氏骨髓－胰臟症候群，簡稱PS或PMPS）為一非常罕見之粒線體疾病，成因為粒線體DNA的大片段刪除。此突變目前大多被認為是在胚胎早期發育中偶發性生成，僅有少數人認為與母系遺傳有關。疾病多在患者嬰兒時期便出現症狀，常伴有骨髓與胰臟功能衰退，同時可能併發可危及性命之乳酸血症與糖尿病。多數患者在嬰兒或幼童時期便會走向死亡階段。本研究中我們診斷了一位七個月大而帶有嚴重貧血、乳酸血症及左眼瞼下垂之Pearson氏症候群患者。透過primer shift PCR、long range PCR與DNA定序等實驗，我們於患者之周邊血單核球細胞DNA中分離出一長達4,237 bp 的粒線體DNA大片段刪除（m.9,486_13,722del），且在刪除點位兩端各帶有一個12 bp長的直接重複序列（5’-TTCGCAGGATTT-3’）。同樣的刪除片段在患者母親之體外受精胚胎滋養層及停止生長之體外受精胚胎中亦有發現，然而在患者雙親的周邊血單核球DNA中皆未偵測到此大片段刪除。綜合上述，因突變發生的一致性使疾病的傳播模式似乎與遺傳有關，卻難以從父母組織當中對下一代的患病率做評估，因此我們認為在高風險家族中，針對Pearson氏症候群的著床前基因診斷是必須的。另一方面，我們也建立了兩株由病人衍生之初級細胞，皮膚纖維母細胞及淋巴球母細胞，本研究中我們亦嘗試建立mitoCRISPR/Cas系統，並成功觀察到細胞表達Cas9信號。未來我們將進一步確認此系統是否能專一性減少含大片段刪除之突變型粒線體DNA，期望能建立有效之mitoCRISPR/Cas基因治療方法。
Pearson syndrome (Pearson Marrow-Pancreas Syndrome, PMPS, PS) is a very rare disease which is caused by a large scale deletion of mitochondrial DNA (mtDNA). The majority consider the mutation to be emerged in early embryonic development sporadically. The others believe it is transmitted by maternal inheritance. The disorder usually begins in infancy, and is characterized by bone marrow and pancreatic failure. Life-threatening lactic acidosis and diabetes may occur too. Most patients die in infancy or early age of childhood. Recently, we have diagnosed the PS patient with critical anemia, lactic acidemia, and left ptosis at seven-month-old. Using long range PCR, primer shift PCR, and DNA sequencing, the 4,237 bp deletion of mtDNA (m.9,486_13,722del) was identified from the patient’s peripheral blood mononuclear cells (PBMCs) with two 12 bp direct repeats (5’-TTCGCAGGATTT-3’) located at the deletion-junction site. Same deletion was further detected in most of the in vitro fertilization (IVF) trophoblasts and degenerated embryos obtained from the mother of the proband. However, no detectable 4,237 bp mtDNA deletion was found in the PBMCs of both parents. Since there seems to be some traits of hereditary transmission of Pearson syndrome, and there are difficulties to evaluate the risk to give birth to babies with the disease from parents’ tissue, we believe the preimplantation genetic diagnosis (PGD) of IVF embryos is crucial for the high-risk families. Furthermore, we also established two patient-derived primary cells, skin fibroblasts and lymphoblasts, looking forward to reducing mutation load of the mutant type mtDNA by gene therapy. Using the mitoCRISPR/Cas system, the mitoCRISPR signal was detected in normal fibroblasts, but no signals were found in control group. We will further address whether the CRISPR-Cas9-mediated mtDNA editing reduce the proportion of 4,237 bp mtDNA deletion in the patient-derived cells. |
