Establishment And Application Of High-throughput Sequencing Platform For Patients With Myeloproliferative Neoplasms

Background and objectivesThe myeloproliferative neoplasms (MPN) are clonal disorders of hematopoiesis characterized by production of mature appearing cells within the blood stream, complication of thrombosis and bleeding, and an ability of developing acute myeloid leukemia (AML) and/or bone marrow fibrosis (MF). In 1960,the Ph chromosome was uncovered in chronic myeloid leukemia (CML), however, research about the genetic of the Ph-negative MPN was little in half a century later. In 2005, the discovery of recurrent somatic mutation JAK2V617F in patients with MPN make the research of MPN into the genome era rapidly. In the next ten years, MPL (TPO receptor)mutation, JAK2 exon 12 mutation and the exon 9 of CALR gene mutation have been found and reported, which provided powerful genetic maker for MPN patients. However, the transformation of different phenotypes of MPN and the ability of developing acute myeloid leukemia (AML) and/or bone marrow fibrosis can’t been explained by a single gene mutation. Some researches reported two or more gene mutation concurrent existed in MPN patients, which give a evidence for the view above. Detecting more molecules, considering various molecular mutation comprehensively, having a further understanding to the pathogenesis of MPN, which gave a help for the clinicians judge prognosis accurately and guide individual therapy.The accurate genetic information of MPN patients was important for clinical research and clinical treatment. For organism, the genome contains the whole of the organism’s genetic information. DNA sequencing can reflect the genetic information of patients accurately, and reveal the complexity and diversity of genetic information in patients. In 1977, the invention of double deoxyribonucleotide end termination (invented by Sanger) and chemical degradation (invented by Gilbert) were markers of the first-generation sequencing technology. At present, Sanger sequencing is still the mainstream method of the DNA sequencing, but the slow speed, the high cost and low-throughput of sequencing impose restriction on application of many field. The next-generation sequencing (NGS) is a high-throughput sequencing technology, which able to detect hundreds of thousands to millions of DNA molecules. The next-generation sequencing with characteristics of quick speed, high accuracy, low cost, deep coverage and high-throughput, allows the researchers to acquire gene position and gene analysis accurately in a short time, which have a extensive application in clinical detection. Ion Torrent PGM platform is a kind of new generation sequencing technology based on semiconductor chip, which direct and real-time detect the ion flow generated in DNA replication through massively parallel semiconductor sensor. Ion Torrent technology has a advantage of high accuracy, quick speed, large flexibility, etc. And it now been applied to many fields of study, such as cancer, hereditary diseases, penetration health field, hematological diseases, microbial and biological basis research, etc.The Thermo Life has launched a Ion Ampliseq scheme, which can amplify thousands of target genes within one single tube and use only 20ng DNA. The Thermo Life has launched Cancer Hotspot commercial Panel, most of the genes related to the solid tumors, gene associated with hematological diseases was little. The scalability of Ion Torrent PGM platform allows researchers to select the target genes of interest and design a customized Panels by Ion AmpliSeq designer. After consulting a large number of reports, we designed a customized MPN Panel, which including JAK2、MPL、CALR、KIT、ASXL1、IDH1、IDH2、LNK、CBL、TET2、 EZH2、DNMT3A、U2AF1、SRSF2、SF3B1、CSF3R、TP53、SOCS1、SOCS2、 SOCS3、NRAS、KRAS and NFE. The purpose of the first part was to evaluate the methodology reliability of the Ion Torrent PGM sequencing platform and the customized Panel in clinical diagnosis of MPN.Based on researches in recent years, about 95%-98% PV (Polycythemia vera) patients,50%-60% ET (Essential thrombocythemia) patients and PMF (Primary myelofibrosis) patients with JAK2 mutation. About 4%-8% ET patients and PMF patients with MPL mutation. CALR mutation was rare in PV patients, which existed in 15%-24% ET patients and 25%-35% PMF patients. However, there were about 15% of MPN patients without the JAK2, CALR and MPL mutation, which were classified as trip-negative MPN. The trip-negative MPN lack the main molecular mutation, which cause it difficult to be diagnosed. The purpose of the second part is to seek the molecular abnormalities and to obtain gene information of trip-negative MPN by next-generation sequencing. So provide a precision diagnosis of MPN for clinicians.Materials and Methodsmaterials1. A total of 10 patients diagnosed with Ph-negative MPN were involved in our study from 2015.01 to 2015.10 in Nanfang Hospital. Sanger sequencing was for 10 patients to detect JAK2V617F and CALR mutation.4 patients with JAK2V617F,4 patients with CALR mutation (2 patients with a 52bp deletion in exon 9,2 patients with a 5bp insertion in exon 9),2 patients concurrent with JAK2V617F and CALR mutation (one with a 52bp deletion of CALR gene, another with a 3bp deletion of CALR gene)2.10 patients diagnosed with Ph-negative MPN were involved in our study from 2015.01 to 2015.10 in Nanfang hospital. Sanger sequencing was for 10 patients to detect JAK2V617F and CALR mutation, none of the 10 patients with JAK2V617F or CALR mutation.6 ET patients,1 CNL patients and 3 PMF patients.Method1. Screening Target gene and designing a customized mulitiplex PCR primers for MPNPrimers for mulitiplex PCR of 23 target genes associated with MPN were designed by Ion AmpliSeq Designer (www.ampliseq.com). All of the 23 genes came from literature.2. DNA extractionExtracting the DNA from bone marrow cells of MPN patients according to the manual operation of DNA extraction kit.3. Sanger sequencingAll of the patients received Sanger sequencing to detect MPL gene.4. Ion Torrent PGM sequencing1) Library PreparationDetecting the concentration of DNA, and the DNA was diluted into lOng/ul with enzyme-free water. Combined customized Ion AmpliSeq panel and Ion AmpliSeq Library kit 2.0 to amplify DNA gene according to the manufacturer’s instructions. Then digesting the primers of the resulting amplifications, adding barcode adapter, purification and quantitative finally.2) Template preparationEmulsion PCR and template-positive enrichment were carried out on Ion OneTouch Two and Ion OneTouch ES respectively according to the manufacturer’s instructions.3) Ion Torrent PGM sequencingThe template of previous preparation was loading on Ion 316 chip, then sequencing was performed on Ion Torrent PGM using the Ion PGM 200 Sequencing Kit. The raw data was stored in the Ion Torrent PGM server and analyzed by ionreporter tool (ionreporter.thermofisher.com).Result1. Mulitiplex PCR PrimersMulitiplex PCR Primers of 23 genes associated with MPN were designed by Ion AmpliSeq Designer, the results as follows:the MPN panel size is 52.42 Kb, a total of 283 amplifications, can cover 97.28% of the target region,283 amplifications packed in two tube,145 amplifications and 138 amplifications respectively, the length of the amplification is between 125 bp and 275 bp.2. Result of Sanger sequencingSanger sequencing for MPL gene of 20 patients with MPN, All of the MPL gene of 20 patients are wide-type.3. Quality control of Ion Torrent PGM sequencingTaking a 316 chip as an example, the IPS Loading was 66%, produced 236 MB raw base data, ISP library enrichment was 100%, polyclonal library and monoclonal library was 33% and 67% respectively. The final monoclonal library was 77% with the exception of test fragments (1%), adapter (12%)and low quality (10%). Usable Reads was 2,159,538. The mean and median read length were 181bp and 193bp respectively. Base reading accuracy in different position was 99.3%, Total bases achieve AQ20 (99%) were 207 Mb. Quality control standards of Ion Torrent PGM sequencing as follows:ISP loading is more than 60%, ISP library enrichment is more than 80%, polyclonal library is less than 40%, the test fragments is less than 5%. The quality control of the chip achieved the Ion Torrent PGM sequencing quality control standards. Taking P1 as an example, Quality control of a single sample as follows:a total of 71,322,697 mapped bases, bases achieve AQ20 (99%) were 64,123,648, the average base coverage is 1302×.4. Result of Ion Torrent PGM sequencing1)10 MPN patients were sequenced by Ion Torrent PGM. In terms of JAK2, MPL and CALR gene mutation.3 patients with the the 52bp deletion of CALR gene are not detected by Ion Torrent PGM sequencing. The rest results of Ion Torrent PGM sequencing are consistence with the Sanger sequencing. Ion Torrent PGM sequencing can display the location of gene, mutation type, change of the amino acids and coverage, the most important is quantify the burden of tumor cells.2) Results of trip-negative MPN by Ion Torrent PGM sequencing as follows:1 ET patients without mutation, the others harboring different numbers of somatic mutations spanning from 1 to 3 variants. TET2 gene mutation, which involved in epigenetic regulation, is the most common mutation in the study.4 patients with the mutation. A CSF3RT618I mutation was detected in the CNL patient, a TP53 mutation was detected in 1 PMF patient. In addition, there are also existed mutations involving spliceosome genes, signal transduction related genes, etc.Conclusion1. In this study, combined the customized mulitiplex PCR primers of 23 gene with the Ion Torrent PGM sequencing platform, successfully developed a good method to detect MPN related gene mutation with characteristics of high sensitivity, specificity, quick speed, and high throughput. Ion Torrent PGM sequencing can quantify the burden of tumor cells, which assisted the clinicians to assess the treatment efficacy.2. The detection of long segment of the deletion by Ion Torrent PGM sequencing were still needed to be further improved.3. In the sduty, nearly all the trip-negative MPN patients exist mutation, mutations of TET2 gene involved in epigenetic regulation was the most common in trip-negative MPN patients. In addition, there are also existed mutations involving spliceosome genes, signal transduction related genes, etc.