Project description
The introduction of Myelodysplastic Syndrome
MDS is a heterogeneous group of hematological malignancies originating from hematopoietic stem cells (HSCs). MDS primarily affects the elderly population and is characterized by hematopoietic abnormalities, various degrees of bone marrow failure, and increased risks of transformation to acute myeloid leukemia (AML)[1,2]. A number of publications indicated that the incidence steeply rises with increasing age and is typically higher among men compared to women [3,4]. The incidence rate and mortality rate of MDS were very low below the age of 60 years but the mortality rate grows rapidly as the age at diagnosis increases (Fig. 1). Irrespective of time period, age standardized incidence-/mortality-rates were almost twice as high among men (3.34-3.55/1.64-2.08 per 100000 py) as among women (1.80-1.82/0.84-0.82 per 100000 py)[5].
Fig. 1 Age-specific incidence and mortality of MDS by sex and period[5] .
Fig. 2. Age-adjusted incidence and mortality of MDS by sex and period[5].
MDS involves progressive development of high co-morbidities with life-threatening conditions. Patients usually have frequent anemia with symptoms that are difficult to treat. Specifically, they are often accompanied by conditions including fatigue, palpitations, shortness of breath, and pale skin. If the patients are in a state of anemia for a long time, it is easy to develop cardiovascular symptoms, such as chest pain, which greatly reduces the patient's life quality. In addition, MDS can lead to a continuous decrease in white blood cells, resulting in low immunity and resistance. As a result, patients are prone to conditions such as skin infections, sinus infections, lung infections. MDS can also cause thrombocytopenia, which leads to reduced platelets and an increased chance of bleeding[6]. A patient in this state may bleed even with a slight bump. The disease progression can be alleviated through active treatment, and the only current cure for this disease is allogeneic hematopoietic stem cell transplantation[7,8].
The diagnosis of MDS
In most cases, those involved in diagnosing MDS are doctors and hematologists. This is because it is often the doctor who identifies anemia during a routine examination, or else MDS is identified on the basis of blood tests carried out to investigate the cause of symptoms of anemia. Once the more frequent causes of anemia have been ruled out, such as iron deficiency, vitamin B12 and folic acid deficiency, and hemolysis, referral to a hematologist for further investigation is advisable. In particular, the presence of bi- or pancytopenia (about 30%) can be a warning signal (red flag) and may indicate bone marrow disease. If blood cell counts and the differential cell count are normal, MDS is extremely unlikely. Patients who have undergone chemotherapy for any other disease, benign or malignant, especially with alkylating drugs (cyclophosphamide, ifosfamide, carmustine, dacarbazine, and others) and/or radiation therapy or radioiodine therapy in the past are at greater risk of developing MDS: around 10% of MDS patients developed the disease after treatment with cytotoxic agents or radiation. Occupational history and any notifications to the employers' liability insurance association appear to be important if there is a possibility that there may have been long-term (many years) exposure to benzole, since this increases the risk of MDS. Once hematological and nonhematological differential diagnoses have been ruled out, careful cytomorphological analysis of blood and bone marrow are necessary, ideally performed by an experienced hematologist or pathologist. It is not unusual, however, for even experienced diagnosticians to fail to make a definite diagnosis, and for this reason repeat bone marrow investigations can sometimes be necessary if the cytopenia persists.
Figure 3. Characteristics of Myelodysplastic Syndromes.[9]
In most cases, MDS is diagnosed by doctors and hematologists, who often identify anemia during a routine examination or perform blood tests to investigate the cause of symptoms of anemia. Once the more frequent causes of anemia such as iron deficiency, vitamin B12, folic acid deficiency, and hemolysis have been ruled out, referral to a hematologist for further investigation is advisable. In particular, the presence of bi- or pancytopenia (about 30%) can be a warning signal (red flag) and may indicate bone marrow disease. If blood cell counts and the differential cell count are normal, MDS is extremely unlikely. Patients who have undergone chemotherapy for any other disease, benign or malignant, especially with alkylating drugs (cyclophosphamide, ifosfamide, carmustine, dacarbazine, and others) and/or radiation therapy or radioiodine therapy in the past are at greater risk of developing MDS: around 10% of MDS patients have developed the disease after treated with cytotoxic agents or radiation. Occupational history and any notifications to the employers' liability insurance association appear to be important if there is a possibility that there may have been long-term (many years) exposure to benzol, which increases the risk of MDS. Once hematological and nonhematological differential diagnoses have been ruled out, careful cytomorphological analysis of blood and bone marrow is necessary, ideally performed by an experienced hematologist or pathologist. It is not unusual, however, for even experienced diagnosticians to fail to make a definite diagnosis, and for this reason, repeat bone marrow investigations can sometimes be necessary if the cytopenia persists.
Due to the difficulty in detecting and treating this disease, many MDS patients globally are delayed in treatment because of the failure to detect MDS in time[10]. Many patients mistakenly believe that they have anemia in the early stage of MDS, thus neglecting the treatment. In addition, the treatment is usually delayed, because MDS is more common in the elderly, who may have psychological fear of hospitals and feudal superstition beliefs. Therefore, it is urgent to develop more effective detection methods for MDS diagnosis. Our team is committed to design a new model based on new biomarkers that can diagnose MDS with specificity, accuracy, and ease in the future.
Dysregulation of RNA splicing and MDS
RNA splicing is an essential event during gene expression in eukaryotes. An accumulation of evidence now suggests that aberrant splicing is strongly associated with many diseases, especially MDS[11,12], leading to the emergence of pharmacological modulation of RNA splicing as a promising therapeutic strategy. There are many RNA splicing-related genes, such as SF3B1, U2AF2, and SRSF2, which have been found to have mutations in around 50% of MDS patients[13,14]. Among these genes, SF3B1 is the most frequently mutated RNA splicing factor in MDS [15], which is present in approximately 25% of MDS patients. Notably, SF3B1 mutations tend to be associated with a good prognosis in MDS [12]. Therefore, the analysis of RNA splicing status could be used for the diagnosis of MDS.
Figure 4. Distribution of recurrent mutations and karyotypic abnormalities in MDS[16].
Clonal cells from ~50% of MDS patients harbor a splicing factor (SF) mutation. Fractions estimated from data in Bejar et al, Papaemmanuil et al, and Haferlach et al.
Our project
According to previous studies, we found that exons of mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and zinc finger protein 91 (ZFN91) were skipped in MDS patients[17,18]. This mechanism gives rise to our idea of constructing two sensors based on the exon skipping of MAP3K7 and ZFN91 to the plasmids containing reporter genes, which monitor the alteration of RNA splicing in cells.Our project will help us to evaluate the effectiveness of these sensors in diagnosing MDS patients in the future.
Figure 5. The dual luciferase reporter system
To measure the alteration of RNA splicing, we engineered two dual luciferase reporter system where two luciferase genes were fused with GTGAGT-recessive exon (MAP3K7 or ZNF91 )-CCACAG minigenes (Fig 4). Upon transfection of the dual luciferase reporters into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing in cells, the internal stop codon would be removed, then the upstream reporter gene (Firefly luciferase, Fluc) and the downstream reporter gene (Renilla luciferase, Rluc) will be placed in the same reading frame to generate a fusion protein Fluc-Rluc. In the case that splicing in inhibited by splicing inhibitors, the stop codon in the recessive exon would lead to the translation termination of the mRNA, thus producing the Fluc protein alone. Therefore, the Fluc gene is expressed regardless of whether splicing occurred, whereas the downstream Rluc gene can only be expressed after splicing. The ratio of Rluc + Fluc to Rluc intensity [(Rluc+Fluc)/Rluc] represents the proportion of spliced transcripts, that is, the splicing efficiency of pre-mRNA.
Figure 6. The GFP-mCherry reporter system
To further monitor the change of RNA splicing in cells, we construed another GFP-mCherry reporter systems where two fluorescent genes were fused with a GTGAGT-recessive exon-CCACAG minigene. A shifted in-frame termination codon (TGATG) was inserted between GFP and mCherry(Fig 5). When the reporter was transfected into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing of the cell, the internally shifted cryptic exon will be removed, mCherry will be expressed. Because the stop codon was located in front of the GFP gene, GFP could not be translatable and red fluorescent could be detected in cells. In the case where splicing is inhibited, the internally shifted recessive exon will not be removed, the mCherry gene is shifted. The GFP is expressed, and the cells show green fluorescence. Therefore, we can monitor the level of splicing in the cell through detecting the different colors of fluorescence.
References:
[1]Greenberg, Peter L., Neal S. Young, and Norbert Gattermann. "Myelodysplastic syndromes." ASH Education Program Book 2002.1 (2002): 136-161.
[2]Cole P, Sateren W, Delzell E. Epidemiologic perspectives on myelodysplastic syndromes and leukemia. Leukemia Res 1995;19:361-5.
[3]Aul C, Gattermann N, Schneider W. Age-related incidence and other epidemiological aspects of myelodysplastic syndromes. Br J Haematol 1992;82: 358-67.
[4]Neukirchen, Judith, et al. "Incidence and prevalence of myelodysplastic syndromes: data from the Düsseldorf MDS-registry." Leukemia research 35.12 (2011): 1591-1596.
[5]Bonadies, Nicolas, et al. "Trends of classification, incidence, mortality, and survival of MDS patients in Switzerland between 2001 and 2012." Cancer epidemiology 46 (2017): 85-92.
[6]Li, W., et al. "Thrombocytopenia in MDS: epidemiology, mechanisms, clinical consequences and novel therapeutic strategies." Leukemia 30.3 (2016): 536-544.
[7]De Witte, Theo, et al. "Allogeneic hematopoietic stem cell transplantation for MDS and CMML: recommendations from an international expert panel." Blood, The Journal of the American Society of Hematology 129.13 (2017): 1753-1762.
[8]Shimoni, A., et al. "Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: the role of dose intensity." Leukemia 20.2 (2006): 322-328.
[9]List, Alan, et al. "Efficacy of lenalidomide in myelodysplastic syndromes." New England Journal of Medicine 352.6 (2005): 549-557.
[10]Tefferi, Ayalew, and James W. Vardiman. "Myelodysplastic syndromes." New England Journal of Medicine 361.19 (2009): 1872-1885.
[11]Mupo, Annalisa, et al. "Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts." Leukemia 31.3 (2017): 720-727.
[12]Malcovati, Luca, et al. "Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms." Blood, The Journal of the American Society of Hematology 118.24 (2011): 6239-6246.
[13]Adès, Lionel, Raphael Itzykson, and Pierre Fenaux. "Myelodysplastic syndromes." The Lancet 383.9936 (2014): 2239-2252.
[14]Hirai, Hisamaru, et al. "A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome." Nature 327.6121 (1987): 430-432.
[15]Hosono, Naoko. "Genetic abnormalities and pathophysiology of MDS." International journal of clinical oncology 24.8 (2019): 885-892.
[16]Bejar, Rafael, and David P. Steensma. "Recent developments in myelodysplastic syndromes." Blood, The Journal of the American Society of Hematology 124.18 (2014): 2793-2803.
[17] Sato, Shintaro, et al. "Essential function for the kinase TAK1 in innate and adaptive immune responses." Nature immunology 6.11 (2005): 1087-1095.
[18]Saminathan, Thangasamy, et al. "Differential gene expression and alternative splicing between diploid and tetraploid watermelon." Journal of experimental botany 66.5 (2015): 1369-1385.