骨髓系增生疾病病人之端粒長度之研究：俱潛力之診斷標的

Abstract

骨髓系增生疾病(Myeloproliferative neoplasms, MPNs)是骨髓系幹細胞發生異常變化使得下游的血液細胞發生過度增生所造成的一種疾病，典型的骨髓增生性疾病病人可分為真性紅血球增多症、血小板增多症和原發性骨髓纖維症。近年來有研究發現在細胞中負責保護染色體末端的端粒，其長度在骨髓增生性疾病病人中有明顯縮短的趨勢。我們藉由一對能結合至端粒重複序列的引子與一對能結合至單一基因(single copy gene)的引子，利用即時監控聚合酶連鎖反應的原理來測量各檢體的相對端粒長度--相對端粒長度(Relative telomere length)正比於端粒產物量(T)比上單一基因產物量(S)之比例(T/S ratio)。 我們分析63 位被測得有高於正常人血球數目的病人其端粒長度並與255 位測有正常血球數目的人做比較，再將此端粒長度的分析結果與其他骨髓增生性疾病突變檢測結果綜合評估。因為在正常人群組中，端粒長度會受個人的性別與年齡影響，因此這些因素在病人中亦被分別分析。 在63 位被測得有高於正常人血球數目的病人中有35 位為JAK2 V617F 陽性的病人。其中27 位V617F 陽性病人的端粒長度較同齡正常人有明顯縮短之現象。此外此端粒長度縮短的現象只有在病人的多核細胞檢體中被發現，在其單核細胞檢體中則否。而在不同性別的V617F 陽性病人比較中，女性病人與男性病人的端粒長度則沒有顯著的差異。在63 位被測得有高於正常人血球數目的病人中剩下的28 位為JAK2 V617F 陰性的病人，藉由甲基特異化聚合酶連鎖反應 (Methylation-specific PCR assay)的結果我們將V617F 陰性的病人分為在JAK2的抑制子SOCS3 promoter 有部份甲基化及無甲基化組。而在SOCS3 promoter有部份甲基化的四位病人中，都可發現其端粒長度都有較同齡正常人下降的表現。 因此實驗認為端粒長度分析可作為檢測JAK2V617F 陰性骨髓增生性疾病中於JAK2 的抑制子SOCS3 promoter 有部份甲基化的病人之另一項標的。

Myeloproliferative neoplasms (MPNs) are caused by several disorders of myeloid clonal stem cells and these diseases are characterized by chronic proliferation of hematopoietic precursors. There are three major types of classic myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and JAK2V617F is the most common biomarker to screen the patients with classic MPNs. Recent studies have reported that telomeres, which are complexes of tandem repeats and able to protect the chromosomes against homologous recombination and non-homologous end joining during cell division, showed significant shortening in patients with MPNs. In this study, the quantitative-PCR assay measured the relative telomere length of 63 studied patients with abnormal increased leukocyte count, or increased platelet count, or increased hemoglobin level; and then the results of the patients was compared with 255 normal controls which have normal complete blood counts (the relative telomere length was the ratio of telomere product to single-copy gene product, T/S). Due to the telomere length was affected by different genders and ages in the normal control cohort, the comparisons of the patients between different genders and ages were processed separately. In the JAK2V617F-positive patient cohort, the results of paired comparisons showed that telomere lengths were remarkably shorter in 27 of 35 patients (27/35) than the age-matched normal controls. The telomere length shortening was observed mainly in the granulocytes but not in the mononuclear cells. Furthermore, no significant differences of telomere shortening were observed between female and maleJAK2V617F-positive patients. In addition, the mutation burden of JAK2V617F displayed no obvious correlation with telomere length shortening in the JAK2V617F-positive patient cohort. In the JAK2V617F-negative patients, abnormal SOCS3 promoter methylation pattern was found in four patients who had significantly shorter telomere lengths than their age-matched normal controls. SOCS3 promoter methylation has been reported among the patients with PMF in the previous reports, so these four SOCS3-positive patients with shorter telomere lengths could have primary myelofibrosis. In conclusion, this data suggested that telomere length analyses may support the diagnoses in patients with SOCS3 promoter methylation but without JAK2V617F.