Changes Of Telomere And Telomerase Activity In Patients With Aplastic Anemia

Background:Telomere is one of the hotspot in life science research in recent years, with the deepening of the research, telomere has gradually aroused the concern of the haematology workers at home and abroad. Short telomere length and telomerase associated gene mutations can lead to hematopoietic stem cell aging, affect the efficiency of hematopoietic stem cells implanted seriously, it is one of the causes of bone marrow failure syndromes.Aplastic anemia is the most common in bone marrow failure syndromes caused by a variety of reasons, mainly characterized by pancytopenia, low bone marrow hematopoietic function, decrease in the number of hematopoietic stem progenitor cells and accompanied by abnormal function. Aplastic anemia’s etiology and pathogenesis is very complex, high heterogeneity, the conventional wisdom is that the mechanism can be summarized as "seed, soil and insect" theory, namely "injury hematopoietic stem cells, hematopoietic microenvironment abnormality and immune dysfunction". In recent years is widely believed that AA is a hematopoietic stem progenitor cells as the target of autoimmune disease, about70%of the patients with immunosuppressive therapy (IST) have improved, but still about30%of the patients invalid and the pathogenesis in those patients may be another reason.In recent years, studies have found aplastic anemia has host genetic susceptibility, Telomeres are short in many patients with aplastic anemia, and mutations affecting telomerase have been identified in these forms of aplastic anemia.Telomerse gene mutations can lead to reduced enzyme activity, accelerated telomere shortening, so hematopoietic progenitor cell proliferation ability decreased. Most of AA patients with telomerase mutations is not sensitive to immunosuppressive therapy.Telomere shortening in patients usually with longer duration of disease, and are hight risk of relapse, clonal evolution,it is more likely progress to advanced malignant clonal disorders, such as transformation of MDS, PNH and AML. So the relationship between telomere, telomerase and aplastic anemia gradually comes to the attention of people.Telomeres are specialized structures of the ends of eukaryon linear chromosomes, consisting of repetitive non-encoding(TTAGGG)n DNA sequences. When they are too short, telomeres signal the arrest of cell proliferation, senescence, and apoptosis.Telomere is not a structural gene, not code for protein, is a highly conserved sequence in evolute cells. Its funetion as an end-Protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation. Telomeres play an important role in maintaining chromosomal integrity.There are two kinds of telomere length regulation mechanism, the main of telomerase and alternative lengthing of telomere(ALT). Telomerase reverse transcriptase (TERT) uses the telomerase RNA component (TERC) as a template to synthesize telomere DNA (TTAGGG) n. The catalytic unit of telomerase contains two copies each of TERT, TERC, and dyskerin, and proteins that stabilize the complex.Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevents the loss of telomeres due to the end replication problems. Prior studies have found that most of the existence of telomerase mutations in patients with aplastic anemia is not sensitive to immunosuppressive therapy clinically, but there is no grouping according to curative effect compare and statistics. Due to mutation detection telomerase positive rate is low, and the high cost, limited it clinical application.Based on the correlation between telomere abnormalities and curative effect of AA, peripheral blood leukocytes or granulocyte was used for specimens in the past, and peripheral blood leukocytes are mainly of mononuclear cells and granulocytes, given the lack of granulocyte in aplastic anemia patients, to get these specimen is not easy, thus this research collect a specimen of peripheral blood mononuclear cells as a source. Select a new method of flow cytometry-fluorescence in situ hybridization (Flow-FISH),which using specific probe telomeres (PNA) to detect the telomere length, and it has good correlation with the usual Southern blot and Q-PCR method, and needed less cell number (<105cell), high sensitivity, can detect telomere length less than3kb, quick operation, strong specificity, repeatability.And use TRAP-PCR-ELISA method for detection of telomerase activity. Objective:Observe the change of telomere length and telomerase activity in patients with aplastic anemia, explore the role of them in pathogenesis, provide a new target of therapy. Observe the relationship between telomere length and curative effect of IST, provides the theory basis for choosing appropriate treatment as soon as possible.Method:(1) We select71cases of aplastic anemia patients between September2010and March2013in General Hospital of Guangzhou Military Command of PLA, Guangzhou First People’s Hospital, etc. Which contains36males and35females, mean age39.48±18.78years (12-82) and35cases of NSAA group,26cases of SAA group,10cases of VSAA group. Collected3ml peripheral blood during the initial diagnosis of disease.At the same time,34cases healthy volunteers with matched age and sex were selected as normal control,17males and17females, mean age40.68±19.00years (10-73). Diagnosis and curative effect of standard reference the Guidelines for the diagnosis and management of aplastic anaemia in2009, this study obtained the consent of the patients themselves or their family members and signed the informed consent form.(2) Testing the relative telomere length in Flow-FISH, cultivate MOLT-4cell lines for internal control cells, specimens mixture with FITC-PNA probe, hybrid, incubation, washing, dyeing.Flow cytometry is used to test the relative telomere length, the result of telomere length in peripheral blood mononuclear cells is in contrast with MOLT-4cell lines as a percentage of average fluorescence intensity value, according to the formula to calculate the relative telomere length (relative telomere length, RTL)=(mean FL1sample cell with FITC-PNA probe-mean FL1sample cell without FITC-PNA probe)×DNA index of controlled cells X100/(mean FL1control cells with FITC-PNA probe-mean FL1in control cells without FITC-PNA probe) DNA index of sample cells. Detect the telomerase activity with TRAP-PCR-ELISA method in patients with aplastic anemia, according to the formula of the relative telomerase activity (RTA)=(AS-ASO)/AS.IS/(ATS8-ATS8.0)/ATS8.IS×100, RTA>0.2for telomerase positive. And analyze the relationship of telomere length and immunosuppressive therapy efficacy.(3) Use SPSS16.0statistical software to analyze, measurement data are expressed in (x±s), multiple factors analysis using linear regression; More comparison using single factor analysis of variance between groups, when the variance is approximate F inspection Welch method.Together using LSD multiple comparison when variance method, the variance is not using Dunnett’T3. Count data to rate, according to comparison between groups by chi-square test or Fisher’s exact probability. Inspection level of alpha=0.05, double side inspection. Results:(1)The groups are divided into normal control group, NSAA and SAA+VSAA group. The linear regression analysis of telomere length and related factors show the telomere length in peripheral blood had no significant difference with different gender (t=0.548, P=0.585), the interaction of gender and diagnosis was not significant (t=-0.580, P=0.563).(2) Peripheral blood telomere length and age showed negative correlation (b=-0.387, P=0.001) in normal control group, NSAA group and SAA+VSAA groups, and telomere length is shorter with the growth of age, and comparison between different diagnosis group, the normal control group telomere length decreased along with the age growth will slightly greater than the other two groups (group NSAA, SAA+VSAA), and NSAA telomeres, SAA+VSAA group trends are basically the same.(3) Control the effect of age on peripheral blood telomere length, using multiple comparison found that NSAA, SAA+VSAA peripheral blood telomere length group were significantly shorter than the normal control group (P<0.001).(4) Selected of23cases in NSAA group,28cases in SAA+VSAA group for telomerase activity, At the same time,34cases healthy volunteers with matched age and sex were selected as normal control,6cases tested were positive in34cases of normal control group (17.6%),11patients were positive in23cases of NSAA group (47.8%),21cases were positive in28cases of SAA+VSAA group (75.0%). Telomerase activity had significant difference (F=20.385, P<0.001).The telomerase activity had no significant difference of age and gender in normal control group, NSAA group, SAA+ VSAA group, using multiple comparison found that SAA+VSAA group (P<0.001), NSAA group (P=0.002) were significantly higher than normal control group. The telomerase activity is2-3times of normal control group.Selected7cases randomly, including2cases of normal control group,5patients with AA, through sequencing method to detect the mutations of telomerase TERT part, and compare the sequence according to the NCBI database data, there are no mutations in them.(5)In NSAA group,the complete response(CR) to the treatment are40.0%(14/35),partial response(PR) to the treatment are45.7%(16/35) the no response(NR) to the treatment are14.3%(5/35).In SAA+VSAA group,CR30.6%(11/36), PR44.4%(16/36), NR25.0%(9/36).(6) Using analysis of variance and chi-square test comparing, curative effect there was no significant difference between patients with different gender (X2=2.3975, P=0.302), the curative effect between NSAA with SAA+VSAA group there was no significant difference (X2=1.489, P=0.475). And age affect curative effect (F=3.138, P=0.05),adjust the effect of age on peripheral blood telomere length, use the linear regression equation, variance analysis found that peripheral blood telomere length has a significant difference between different therapeutic group and control group (F=4.615, P=0.003), multiple comparison found that telomere length in NR group was significantly lower than normal control group (P=0.005), telomere length in CR/PR group compared with normal control group had no significant difference (P=0.517, P=0.254). When telomere length is below29.21,IST curative effect will significantly influence, that is invalid will increased obviously.Conclusion:Telomere length of peripheral blood mononuclear cell in patients with Aplastic anemia was significantly shortened with normal control group, it can directly lead to bone marrow failure, telomerase activity in aplastic anemia patients not low,but rise instead, telomere length was significantly lower than normal control group in unsensitivity to immunosuppressive therapy group, this part of the telomere and telomerase abnormality in patients with aplastic anemia are different in pathogenesis, disease progression, treatment, etc.It is necessary to detect of telomere length in peripheral blood cell, telomerase activity and the relevant mutation, adjust the treatment plan.Telomere and telomerase may become the new targets of treatment in aplastic anemia.