| Background and ObjectivesAlpha-thalassemia is one of the most common and influential human health monogenic diseases, prevalent not only in the countries of the Mediteranian seacoast, but also in the several Africa regions, South East Asia and Southern China. It is conservatively estimated that there are nearly 200 million carriers worldwide. In the southern regions of China, such as Guangxi, Guangdong and Sichuan, the carrier rates of population are 17.55%,8.53%and 1.92%respectively. At present, there are no effective treat measures for the Thalassemia. Then, the first selected precautionary measure is to prevent the birth of severe thalassemia child by population screening and prenatal diagnosis with coresponding analytical technique. For the wide distribution and severity of thalassemia, the precautionary measures that aim to lower the birth rate of severe thalassemia child are successively carried out by the thalassemia affected countries and regions in the world.The human a-globin gene cluster is located at chromosome 16. The cluster is made up of three functional genes such as ζ, al and a2 that express ζ-globin chain and a-globin chain which combine with β-globin to form ζΔγ2 (Hb Portland in embryonic period) and β2β2 (adult hemoglobin) respectively. Under normal conditions, the a and β globin chains expressed from human globin genes have proper ratio to form functional hemoglobin tetramer. It is the molecular mechanism of alpha-thalassemia that theβ-globin chains are relative surplus result from the a-globin chain decreased synthesis caused by a-globin gene defects. The a-globin gene deletion is the main(>95%) reason that lead to a-globin chains less synthesis, and the clinical severity of a-thalassemia is directly proportional to the number of a-globin genes affected.In clinical practice of alpha thalassemia molecular diagnosis, the Southern blotting is accurate, but this method is incompatible with routine detection for the tedious-operation, time-consuming, labor-intensive and low-throughput. Being widely applied at present, the gap-PCR method needs to detect every known deletion type step by step. So it is heavy workload and low-throughput, as well as the false negative caused by missing of unknown new deletion type and false positive result from the exogenous contamination. Recently, though a few new techniques have been applied and some advances have been achieved, the alpha-thalassemia molecule diagnostic method is also based on the gap-PCR technique and has no substantive breakthrough. The operational feasibility and detection throughput of the diagnostic methods could not meet the need of large-scale population screening and routine examination.The pathogenesy of alpha-thalassemia is the deletion of a-globin genes, so the ultimate purpose of any diagnostic method is to determine the deletion. Gap-PCR is the technique that based on estimation of functional gene deletion by qualitative examination of deletion one by one. So it is inevitable that have bottlenecks of false negative and false positive, as well as the difficulty to realize the large scale detection with high throughput. With the implementation of thalassemia prevention project and the research development of thalassemia molecular mechanism and epidemiology, a high throughput diagnostic method with accuracy, reliability, simplicity, automation and standardization realizable, suitable for large-scale population screening and routine diagnosis, is the urgent need of public medical for large-scale alpha thalassemia population screening, and also is the new challenge of diagnostic method for thalassemia.The objective of this study is to establish a new molecular diagnosis pathway and method for alpha thalassemia diagnosis, effectively resolve the bottlenecks existed at present such as false negative and false positive. The new molecular method is accurate, reliable, simple, practical, high-throughput with low cost, and meet the current method need of large scale population screening and routine diagnosis for alpha-thalassemia.Programs and methodsAccording to the molecular basis of a-thalassemia, it will be able to accurately diagnose a-thalassemia if the deletion number of a-globin genes can accurately analyze. As far as the human normal genome concerned, the genotype of a-globin is ζ2/α/α2/a22, has two copies respectively. Some HKGs, such as GAPDH, has no copies number variation with two copies under any circumstance. Then the copies of a-globin gene and house-keeping gene are equal in a normal gDNA sample. If one of the a-globin gene deleted, then the copy number of this globin gene is only half of HKG’s, otherwise, if one of the a-globin gene has three copies, it’s copy number is 1.5 times of HKG’s. Therefore, as long as this ratio of copies number is accurately detected with appropriate techniques, the a-globin gene deletions can be accurately analyzed, and then the quick molecular diagnosis of alpha thalassemia can be realized. Based on this research strategy, the research programs and methods have been carried out in this study are as follow.①The relative copies number of ζ, αl and α2 gene were analyzed by real-time fluorescence quantitative PCR with the △Ct value relative quantitation method that the GAPDH gene was selected as reference gene while the ζ, αl and α2 genes were selected as target genes. The real-time fluorescence PCR technology can precisely quantify the copies of samples’DNA sequence.The theory is based on the log-linear relation of Ct value and template initial copies, so the more the initial copies, and the less Ct value. As for a normal specimen, the copies number of a-globin gene and GAPDH is equal, and then the difference of Ct value (△Ct) between them is constancy. If one is a thalassemia carrier that one of the a globin gene deleted, the a globin gene copies decreased 1 time and the Ct value increased 1, then the △Ct also increased 1 and the 2-△△Ct value is 0.5. From these data, the 2-△△Ct value demonstrated that the relative copies ratio between target gene and reference gene is 0.5. Based on the theory and the principle of data analyze, the target gene deletions can be accuratly analyzed by this relative quantitaion method.2. Establish a quadruple Taqman real-time fluorescence quantitative PCR system which can realize the simultaneously relative copies analyze of ζ, αl and α2 globin genes. The △Ct relative quantitative manner selected in this study is based on the copies ratio of target and reference gene which in the same volume and concentration of gDNA sample. In order to eliminate the Ct value error result from the difference of templete amount under the single tube for single gene detection, a quadruple PCR system is established in this study. The target and reference gene are detected with same added templete amount and amplification condition. Otherwise, in order to ensure high specificity and high amplificaion efficiency of quadruple PCR system, a templete preamplification treatment measure that the detection templete is synthesized with lower annealing temperature and subsequently detect the templete with high annealing temperature is used.3. The overall optimization of the quadruple Taqman real-time fluorescence quantitative PCR system. According to relate papers and the needs of the △Ct value relative quantitation with 2-△△Ct date analysis, the amplification efficiency of target and reference gene must to be high and basically the same. After the specificity verification of primers of GAPDH,ζ,αl and α2 gene, the ingredients of this quadruple PCR system such as concentration of primers, probes, PCR Buffer, Mg2+ and templete amount, were Systematically optimized and adjusted. The optimal quadruple Taqman real-time fluorescence quantitative PCR system was decided according to the results of amplification efficiency and consistency assessment.4. The sensitivity and accuracy evaluation of the new diagnostic method applied in deletional alpha-thalassemia molecular diagnosis. The laboratory molecular diagnostic method for genetic disorder is aimed at reaching the expected diagnosis objective by applying certain detection system. So the optimization of detection system is prerequisite, while the evaluation of sinsitivity and accuracy is crucial in the progress of molecular diagnostic method setting up. At first,12 gDNA samples of known genetypes that detected by gap-PCR and verificated by Southern Blot were selected, and the relative copies of ζ, αl and α2 that derived from the deletions were set as target value. Then, the results of relative copies that obtained from the detection of these samples with new detection system were compared with the target value. Subsequently,218 gDNA samples of known genetypes that detected by gap-PCR were also detected in the same way. The sensitivity and accuracy of this new method applied in deletional alpha-thalassemia molecular diagnosis were overall assessed by the way of these 230 known genetypes samples detection.5. The preliminary evaluation of the new diagnostic method in deletional of a-Thalassemia in population screening. To provide a simple and pragmatic molecular diagnostic method for currently population screening for deletional alpha-thalassemia is one of the objectives in this study. In order to evaluate the practicality and feasibility of the new diagnostic methods used in population screening for deletional a-Thalassemia,104 gDNA samples of random population were collected. The samples were detected simultaneously with the new diagnostic method and gap-PCR. Then the results were compared and the concrete situations of experiment procedure were also analyzed.6. The evaluation of the new diagnostic method applied in large sample blind analysis. 546 gDNA samples that the phenotypic information available, the concentration 100-300ng/μl,the OD260/280>1.55, the common deletional of alpha-thalassemia detected with gap-PCR, were numbed blindly, and then detected with this new diagnostic method. The results of relative copies were compared with that derived from the gap-PCR genetype. The gap-PCR and/or MLPA were used to redetect the inconsistent sample. The sensitivity, accuracy and practicality of this new diagnostic method applied in deletional a-Thalassemia and a-globin gene CNVs molecular diagnosis were overall assessed by this way.ResultsAccording to the study objectives and research strategies, the obtained results from the implementation of concrete study schedules and process are list as follow:1. Established a stable and reliable quadruple TaqMan real-time quantitative fluorescent PCR system. In this system, the specificity of the selected amplifying DNA target sequences and the designed primers were proved to be reliable. The Measures of template pre-treatment is practicable.2. A quadruple TaqMan real-time quantitative fluorescent PCR optimized system was established. After a series of optimization and adjustment of the PCR reaction system and condition, as well as the evaluation of amplification efficiency and consistence, the optimized system is that a total reaction volume of 20μl which contains 1.5 x PCR Buffer,7mmol/L Mg2+,75nmol/L per pair of primers, 100nmol/L per strip of probes, lu Ex Taq enzyme,100-300ng gDNA,0.2mmol/L dNTPs. The PCR amplification parameters were 95 ℃ pre-denaturing 5 minutes, 94℃ 30sec and 52℃ 20sec for 3 cycles,92 ℃ 10sec and 62 ℃ 45sec with the fluorescent signal detected for 32 cycles. Under such optimum conditions, the amplification efficiency of ζ αl, α2 and GAPDH gene were 101.8%,102.8%, 102.8% and 102.0% respectively, which all close to 100%. The analysis-curve slopes of the amplification efficiency consistency evaluation of ζ, αl and α2 target genes were 0.0005,0.0013 and 0.0032 respectively, which all close to 0. These results demonstrated that the amplification efficiencies of this multi-system were high and basically identical, which meet the requirements of 2-△△Ct relative quantification.3. The sensitivity and accuracy of this new method for deletional a-thalassemia diagnosis all reached 100%. The relative gene copies of alpha globin gene that obtain from the detection of the 230(12+218) known genetype gDNA samples which detected with gap-PCR and/or Southern Blot.4. The new diagnostic method is simple operation, quickly detection, stable and reliable results, and suitable for deletional alpha-thalassemia population screening. In the preliminary evaluation of deletional α-thalassemia population screening,104 random population gDNA samples were simultaneously detected with this new method and gap-PCR.16 deletional α-thalassemia were detected respectively and the results were consistent each other between these two methods. These 16 samples were all the common deletional of local region. On the procedure of detection, this new method was simple, easy-to-conduct, high -efficiency and high-throughput, as well as the detection results is accurate and reliable. These results demonstrate the sensitivity and specify of this new method is all 100%, and also gave a preliminary demonstration of feasibility and practicality of this new method applied in large scale population screening.5. The new method can not only realize the rapid molecular diagnosis of deletional of α-Thalassemia, but also effectively resolve the technical bottlenecks such as false-negative and false-positive. According to the analysis results of the 545 blind samples, all samples of common deletional alpha-thalassemia were detected and were in line with gap-PCR. Furthermore,2 samples of rare deletional with negative gap-PCR results were detected by MLPA,2 samples of --3.7/αα contaminated by--SEA with positive gap-PCR results were detected and verified by phenotypic data analysis and MLPA. At the same time, a few samples with al or a2 gene multiple copies were detected. The evaluation results show that this new diagnostic method can realize the rapid molecular diagnosis for deletional alpha-Thalassemia with 100% sensitivity and specify, and can effectively resolve the false-negatives and false-positive misdiagnosis. Furthermore, though the sensitivity and specify need to be improved, this method can detection α-globin gene CNVs.ConclusionThrough this study, a new molecular diagnosis way for deletional alpha-thalassemia that based on target gene copies number direct quantification is established in theory aspect, and a new molecular diagnostic method for deletional alpha-thalassemia that based on quadruple TaqMan real-time quantitative fluorescent PCR system is also constructed in methodology aspect. This new molecular diagnosis way and method, was directly analyze the relative copies number of ζ,αl and α2 globin gene with quantitative mode, then effectively avoid the false negative that result from the genetic heterogeneity of deletional. The utilization of TaqMan real-time quantitative fluorescent PCR no only effectively eliminate the false positive caused by small amount of foreign-source contamination, bust also make it easy to realize automation and standardization. Otherwise, the analysis of gene deletion and CNVs can be implemented simultaneously, then the workload, testing costs and test time can be greatly reduced while the detection throughput and efficiency improved.Through the overall evaluation of sensitivity and accuracy, as well as the preliminary assessment of the practicality in population screening, it can be fully demonstrated that the new molecular diagnostic method established in this study is accurate, reliable, simple, practical and high-throughput with low-cost, suit for alpha thalassemia large-scale population screening and routine diagnosis. Moreover, the deletional alpha-thalassemia is the typical type of gene deletion genetic disorders. The molecular diagnostic ways and methods established in this study have universal representaion and versatility, which can provide reference for molecular diagnostic method research of other gene deletion genetic disorders.Background and ObjectiveHuman ζ-globin protein, which constitutes the subunits of embryonic hemoglobin during human development is encoded by ζ-globin gene on chromosome 16 and participates in forming Hb Gowerl (ζ2ζ2) and Hb Portland I (ζ2γ2).At the eighth week of embryonic stage, there is a switch in expression of the human β-like globin gene cluster from the ζ globin gene to a globin genes[1]. It has been found that, when there are gross deletions of the human a-globin gene cluster the expression of ζ-globin gene will delay, namely that ζ globin appears in a certain level during fetal life and adulthood when the expression of ζ globin gene should be closed. This unique phenomenon currently in the world can be encountered in only three types of deletion thalassemia-Southeast Asia deletion alpha-thalassemia (--SEA/αα), Mediterranean deletion alpha-thalassemia (--MED/αα) and Spain deletion alpha-thalassemia (--SPAN/αα) [2-3]. Due to ethnic differences, the last two types are relatively less, and only happen in Europe crowd. Southeast Asia deletion alpha-thalassemia (--SEA/αα) is the main thalassemia type in Southeast Asia and Southern China [4-6] Then it was reported that the micro ζ-globin in the blood was detected with radio immunoassay (RIA) to screen SEA deletion alpha-thalassemia[7]; The United States UBI MAGIWEL established quickly and convenient alpha-thalassemia (-SEA/αα) diagnostic enzyme-linked immunosorbent assay (ELISA) witζ-globin as specific genetic marker in 2002[8]; We also established SEA deletion alpha-thalassemia ELISA kit for detection ofζglobin chain in 2009 [9].The related studies of the regulation mechanism of globin gene expression have been emphasized, and the molecular basis influencing the amount of globin expression can be used as an important reference for genetic diagnosis of thalassemia [10-12].Our data obtained in the development ofζ-globin qualitative kit have indicated that theζ-globin expression is different among different individuals of the same genotype as well as SEA carriers of different genotypes, such as --SEA/αα,--SEA/-α and-SEA/αTα. Study on the specific molecular mechanism leading to this expression difference has an important significance in understanding the regulation of globin gene expression. Thus, establishing a simple, accurate and reliableζ-globin quantitative detection system, is the prerequisite to realize the foregoing scientific research.The purpose of this study were with SEA deletion alpha-thalassemia ELISA kit for detection ofζ-globin chain, developed by us, as the basic detection system [9], to determine the effective concentration range of quantitative detection by prepared ζ-globin standard, develop aζ-globin double antibody sandwich ELISA quantitative detection system, evaluate its specificity, anti-interference ability and stability, and then apply it to theζ-globin quantitative detection of fetal amniotic fluid, prenatal and newborn umbilical cord blood and adult peripheral blood of normal population and SEA deletion alpha-thalassemia carriers in this region, providing methodological support and accumulating the relevant data for the next research. The research mainly included the following aspects:Methods and results1 The purification and identification of human naturalζ-globinAffinity chromatography column was prepared with anti-ζ-globin monoclonal antibody as ligand, to separate naturalζ-globin from the umbilical cord blood hemolysate of Hb Bart’s edema fetus. Analyzed by SDS-PAGE, the separated natural ζ-globin was a mixture of two proteins with molecular weight of 16KD and 32KD, which were confirmed by Western Blot to be ζ-globin monomer and ζ-γ globin dimer respectively. Subsequently, gel filtration was used to purifyζ-globin monomer from the mixture. The molecular weight of ζ-globin monomer was identified to be 16KD (purity>>99.0%) by SDS-PAGE, Western Blot and HPLC, followed by ELISA, the result of which showed that the antigenicity was reliable, the natural biological activity was maintained, and the epitops were the same as or similar to reorganized ζ-globin. Finally, the protein concentration of the purified natural ζ-globin monomer, which was determined using the reference method, was defined as the standard reference materials of quantitative detection.2. The establishment of ζ-globin double antibody sandwich ELISA quantitative detection systemOn the basis of the purified natural ζ-globin monomer standard reference materials, with SEA deletion alpha-thalassemia ELISA kit for detection ofζ-globin chain, developed by us, as the basic detection system, the quantitative detection effective concentration range was determined to be 0.5-12μg/ml based on the standard materials concentration gradient absorbency linear region, and was followed by the preparation of the Hb Bart’s edema fetus umbilical cord blood hemolysate which contained naturalζ-globin. Standardized on the purified natural ζ-globin monomer standard reference materials, the quantitative ELISA method in combination with the method of specimen gradient dilution were employed to certify the concentration ofζ-globin in hemolysate, and thereby the certified hemolysate was used as the standard in the routine quantitative detection system.3. The assessment of ζ-globin double antibody sandwich ELISA quantitative detection systemBy adding interference to the purified natural ζ-globin monomer standard reference material of known concentration, a recycling experiment was performed, which suggested inorganic salt, plasma protein and other types of hemoglobin contained in routine specimens, and Sodium azide preservatives with a concentration of no more than 0.03% did not affect the quantitative test results. The standard was frozen and kept refrigerated to appraise the storage stability and detection repeatability of repeated freezing and thawing specimens, and the results showed that preserved at 4 ℃ within seven daysζ-globin’s antigenicity had no apparent change, at-20 ℃ with no repeated freezing and thawing ζ-globin can be kept for a long time, and at-20 ℃ with repeated freezing and thawing over 3 times ζ-globin’s antigenicity reduced greatly. At the same time, quantitatively testing the ζ-globin in the ζ-γ globin dimers showed that dimers did not affect the quantitative test results.4.ζ-globin double antibody sandwich ELISA quantitative detection of clinical samplesUsing this quantitative detection system established by us, amniotic fluid ζ-globin quantitative detection of 60 normal, --SEA/αα and Hb Bart’s fetal showed ζ-globin was not detected in amniotic fluid samples of the three genotypes. Umbilical cord bloodζ-globin quantitative detection results of 85 normal and --SEA/αα fetal demonstrated there wasζ-globin in fetal life of these two genotypes, the expression lecreased with the increase of gestational age, and the amount of --SEA/aa fetal ζ-globin was significantly higher than normal fetal. Peripheral bloodζ-globin quantitative analysis of 71 normal individuals of every age group showed that in normal people of 3 months after birth there were still a fewζ-globin chains. Peripheral bloodζ-globin quantitative analysis of 278-SEA/ααinfants and adult indicated that, in --EA/aa crowd, there was obviousζ-globin expression difference, with nearly 14% --SEA/αα individuals expressingζ-globin high. As the results of peripheral bloodζ-globin quantitative analysis of 68 HbH patients showed, HbH patients had significantly lowerζ-globin expression than --SEA/αα.5. Further researchThe next step research work will focus on further improve the quantitative detection system, promote the application of ζ-globin detection in basic research and clinical work. Meanwhile, in the light of the difference of ζ-globin expression in different genotypes and different individuals, discuss the molecular mechanisms, and explore the gene regulation.ConclusionsOn the basis of SEA alpha-thalassemiaζ-globin chain qualitative testing system, this study makes use of affinity chromatography and gel filtration to purify natural ζ-globin monomer as standard reference materials, based on which hemolysate that contains natural ζ-globin is certified. Defining this hemolysate as the routine detection standard, we successfully establish human ζ-globin ELISA quantitative detection system with the detection concentration range of 0.5-12μg/ml. The quantitative test system described here is a simple, stable and reliable assay that is adaptable for application in ζ-globin quantitative detection of fetal amniotic fluid, prenatal and newborn umbilical cord blood and adult peripheral blood. |
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