摘要
目的 探讨BCR-ABL1阴性骨髓增殖性肿瘤(MPN)钙网蛋白(CALR)基因突变分类及其与临床表现的关系.方法 采用基因组DNA聚合酶链反应(PCR)扩增产物Sanger测序法检测2015年11月至2018年11月上海交通大学医学院附属瑞金医院236例BCR-ABL1阴性MPN患者(除外真性红细胞增多症及CALR突变阴性)CALR基因第9号外显子突变情况,根据PCR测序结果,分为52 bp缺失(1型)突变、5 bp插入(2型)突变及其他类型突变,比较原发性血小板增多症(ET)(198例)和原发性骨髓纤维化(PMF)(38例)患者间两种突变携带者的临床特征.对测序法无法分类的突变类型根据突变蛋白肽链α螺旋倾向分值和带负电荷氨基酸残基保留程度进行分类,比较两种分类方法的差异.结果 236例患者中,206例(87.3%)检测出CALR基因1型或2型突变,包括ET 173例(99例1型突变,74例2型突变),PMF 33例(28例1型突变,5例2型突变);30例为其他类型CALR基因突变,包括ET 25例,PMF 5例. 173例CALR基因突变ET患者中,1型突变患者白细胞计数高于2型突变患者[(8.6±2.7)×10^9/L比(7.6±2.4)×10^9/L,t=2.45,P=0.015];33例CALR基因突变PMF患者中, 1型突变患者年龄高于2型突变患者[(58±13)岁比(41±16)岁,t=2.51,P=0.018].根据突变蛋白肽链α螺旋倾向分值和肽链带负电荷氨基酸残基保留程度对测序法非1型和非2型27种突变分类时,二种方法有差异.根据突变蛋白肽链α螺旋倾向分值分类时,PMF患者中1型+1型样突变比例高于其在ET患者中比例[78.9%(30/38)比56.6%(112/198),P<0.01];根据突变蛋白肽链带负电荷氨基酸残基保留程度分类,突变蛋白肽链的等电点(pI)值明显高于野生型序列,1型样突变蛋白肽链的pI值高于2型样突变(11.79±0.15比10.02±0.42,t=11.51,P<0.01).结论 CALR基因1型突变的ET患者可能与骨髓纤维化转化的高风险密切相关.根据突变蛋白肽链α螺旋倾向分值和肽链带负电荷氨基酸残基保留程度对CALR突变分类的结果存在差异,需要对CALR突变导致MPN发病机制进一步研究,以明确突变类型与疾病预后的关系.
Objective To investigate calreticulin (CALR) gene mutations classification in BCR-ABL1 negative myeloproliterative neoplasms (MPN), and its relationship with clinical manifestations. Methods Genomic DNA polymerase chain reaction (PCR) amplification product Sanger sequencing method was used to detect the mutation of exon 9 of CALR gene in 236 patients with BCR-ABL1 negative MPN (excluding polycythemia vera and negative CALR mutations) in Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from November 2015 to November 2018. The mutations were classified into 52 bp deletion (type 1) mutation, 5 bp insertion (type 2) mutation and other mutation types according to PCR sequencing analysis. The clinical characteristics of the carriers with two kinds of mutations in 198 patients with essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) were compared. For the types of mutations that could not be determined, they were classified according to the α-helix propensity score of the mutant protein peptide chain or the degree of retention of the negatively charged amino acid residues, and the differences between the two classification methods were also compared. Results Among 236 patients, the CALR gene type 1 or type 2 mutation was detected in 206 cases (87.3%), including 173 ET patients (99 cases of type 1 mutation and 74 cases of type 2 mutation) and 33 PMF patients (28 cases of type 1 mutation and 5 cases of type 2 mutation). The CALR non-type 1 or non-type 2 mutation was detected in 30 cases, including 25 ET patients and 5 PMF patients. Among 173 ET patients with CALR gene mutation, the white blood cell count (WBC) of patients with type 1 mutation was higher than that of patients with type 2 mutation [(8.6±2.7)×10^9/L vs. (7.6±2.4)×10^9/L, t= 2.45, P= 0.015]. Among 33 PMF patients with CALR gene mutation, the age of patients with type 1 mutation was older than that of patients with type 2 mutation [(58±13) years old vs. (41± 16) years old, t=2.51, P=0.018]. According to the α-helix propensity score of mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues, 27 kinds of non-type 1 or non-type 2 mutations were classified by using sequencing method, and there were differences between the two methods. According to the α-helix propensity score of the mutant protein peptide chain, the proportion of type 1/type 1-like mutation in PMF patients was higher than that in ET patients [78.9% (30/38) vs. 56.6% (112/198), P <0.01]. According to the degree of retention of negatively charged amino acid residues in the mutant protein peptide chain, the isoelectric point (pI) value of the mutant protein peptide chain was higher than that of the wild type sequence. The pI value of the type 1-like mutant protein peptide chain was higher than that of the type 2-like mutation (11.79±0.15 vs. 10.02±0.42, t= 11.51, P < 0.01). Conclusions Type 1 mutated ET patients may be closely related to the high risk of myelofibrosis transformation. The results of the classification of CALR mutations are different according to the α-helix propensity score of the mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues. Further study is necessary to identify the pathogenesis of MPN caused by CALR mutation, and to determine the relationship between mutation type and prognosis of disease.
作者
陆一一
林琳
王学锋
Lu Yiyi;Lin Lin;Wang Xuefeng(Department of Clinical Laboratory,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200020,China)
出处
《白血病．淋巴瘤》
CAS
2019年第11期641-646,共6页
Journal of Leukemia & Lymphoma
