RESUMO
Epitranscriptomics analyze the biochemical modifications borne by RNA and their downstream influence. From this point of view, epitranscriptomics represent a new layer for the control of genetic information and can affect a variety of molecular processes including the cell cycle and the differentiation. In physiological conditions, hematopoiesis is a tightly regulated process that produces differentiated blood cells starting from hematopoietic stem cells. Alteration of this process can occur at different levels in the pathway that leads from the genetic information to the phenotypic manifestation producing malignant hematopoiesis. This review focuses on the role of epitranscriptomic events that are known to be implicated in normal and malignant hematopoiesis, opening a new pathophysiological and therapeutic scenario. Moreover, an evolutionary vision of this mechanism will be provided.
Assuntos
Epigênese Genética , Neoplasias Hematológicas/metabolismo , Hematopoese , Transcriptoma , Adenosina Desaminase/genética , Animais , Epigenômica , Evolução Molecular , Humanos , Transferases Intramoleculares/genética , Mutação , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genéticaRESUMO
We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.
Assuntos
Análise Mutacional de DNA , Proteínas de Fusão bcr-abl/genética , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Fusão bcr-abl/sangue , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mutação , Nanoporos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Mixed phenotype acute leukemias (MPAL) include acute leukemias with blasts that express antigens of more than one lineage, with no clear evidence of myeloid or lymphoid lineage differentiation. T/myeloid (T/My) MPAL not otherwise specified (NOS) is a rare leukemia that expresses both T and myeloid antigens, accounting for less than 1% of all leukemias but 89% of T/My MPAL. From a molecular point of view, very limited data are available on T/My MPAL NOS. CASE PRESENTATION: In this report we describe a T/My MPAL NOS case with a complex rearrangement involving chromosomes 5 and 14, resulting in overexpression of the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) gene due to its juxtaposition to the T cell receptor delta (TRD) gene segment. CONCLUSION: Detailed molecular cytogenetic characterization of the complex rearrangement in the reported T/My MPAL case allowed us to observe ADAMTS2 gene overexpression, identifying a molecular marker that may be useful for monitoring minimal residual disease. To our knowledge, this is the first evidence of gene dysregulation due to a chromosomal rearrangement in T/My MPAL NOS.
Assuntos
Proteínas ADAM/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 5/genética , Leucemia Aguda Bifenotípica/genética , Pró-Colágeno N-Endopeptidase/genética , Translocação Genética , Proteínas ADAMTS , Proteína ADAMTS4 , Adolescente , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Aguda Bifenotípica/patologia , Masculino , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
BACKGROUND: The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5-10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified. FINDINGS: In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed. CONCLUSIONS: Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Translocação Genética , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Análise por Conglomerados , Redes Reguladoras de Genes , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfotransferases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
Inflammatory bowel diseases (IBDs) are a group of chronic conditions of the gastrointestinal tract in which nationwide studies have revealed a higher risk of hematological malignancies (HMs). Clonal hematopoiesis (CH) is a premalignant condition defined by the presence of an acquired somatic mutation characterized by a variant allele frequency (VAF) of ≥2%, in a gene frequently associated with HMs. A growing body of evidence suggests a correlation between inflammation and CH; its occurrence in the context of IBD has been previously demonstrated. With the aim to assess CH possible co-occurrence in patients with an IBD associated with HMs, we performed a targeted next-generation sequencing analysis in a cohort of thirteen patients who were referred to our center with IBD associated with HMs. Eleven (85%) patients showed one or more mutations in CH-associated genes; DNMT3A was the most frequently mutated gene, followed by ASXL1 and JAK2. These results may suggest that the mechanisms at the basis of the inflammatory environment could potentially select for the growth of hematopoietic clones harboring specific mutations. In this context, CH emergence may be boosted by the proinflammatory IBD environment, thus acting as a biological link between IBD and the HM onset. If these data are confirmed, IBD patients screened and positive for CH should undergo a hematologic follow-up to assess the risk of developing HM. Future study will clarify the relationship between these conditions.
RESUMO
The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.
Assuntos
Sequência de Bases/genética , Cromossomos Humanos Par 7/genética , Proteínas de Fusão bcr-abl/genética , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Deleção de Sequência , Adolescente , Adulto , Idoso , Crise Blástica/genética , Crise Blástica/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 7/metabolismo , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Estudos de Coortes , Éxons/genética , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Fator de Transcrição Ikaros/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoAssuntos
Calreticulina/genética , Mutação , Transtornos Mieloproliferativos/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Análise Mutacional de DNA , Frequência do Gene , Humanos , Transtornos Mieloproliferativos/diagnóstico , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genéticaRESUMO
The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude.
Assuntos
Genes de Imunoglobulinas , Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Sequenciamento por Nanoporos , Análise Mutacional de DNA , Humanos , Região Variável de Imunoglobulina/genéticaRESUMO
Acute promyelocytic leukemia (APL) patients carry in 27% of cases an activating mutation of the fms-like tyrosine kinase-3 (FLT3) gene: internal tandem duplication (ITD) or tyrosine kinase domain (TKD) point mutation. The simultaneous presence of both types of mutations, so-called FLT3 dual mutations, has been reported in 2% of APL, but this circumstance has never been studied. We studied a cohort of 74 APL cases, performing an in-depth analysis of three FLT3 dual mutant cases. Nanopore sequencing (NS) allowed us to characterize their complex mutational profile, showing the occurrence of multiple activating FLT3 mutations on different alleles in the leukemic promyelocytes and suggesting a cumulative impact of these events on the constitutive activation of the FLT3 pathway in APL cells. NS approach not only sheds light on the FLT3 mutational complexity in APL, but may also be useful to better clarify the FLT3 mutations landscape in acute myeloid leukemia.
Assuntos
Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Sequenciamento por Nanoporos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/genética , Mutação , Tirosina Quinase 3 Semelhante a fms/genéticaAssuntos
Proteínas de Ciclo Celular/genética , Cromossomos Humanos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX5/genética , Proteínas/genética , Aberrações Cromossômicas , Proteínas do Citoesqueleto , Humanos , Hibridização in Situ Fluorescente , Proteínas de Ligação a Poli-ADP-Ribose , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Prognóstico , Fatores de Transcrição , Translocação Genética/genética , Ubiquitina-Proteína LigasesRESUMO
We report on a boy with three cell lines: 46,XY, r(11)(p15.5,q25)[90]/45,XY,-11 [8]/47,XY, r(11)(p15.5,q25)x2[2], with minor anomalies and mental retardation who developed asynchronous bilateral Wilms tumors (WTs). Array comparative genomic hybridization (CGH) performed on peripheral blood leukocytes of the patient led to the identification of a constitutional duplication of 4.8 Mb at 11p15.5-11p15.4. This duplication was found to involve the chromosome of paternal origin, and occurred in tandem on the ring chromosome 11. Despite the constitutive duplication of the paternal 11p15 chromosome region, the patient showed no sign of Beckwith-Wiedemann syndrome. However, the molecular characterization of the two neoplasias was consistent with their independent origin and showed that they arose from the two distinct cellular clones with the ring chromosome, indicating that this anomaly is likely to have caused the patient's susceptibility to WT development.
Assuntos
Cromossomos Humanos Par 11/genética , Análise Citogenética , Mosaicismo , Cromossomos em Anel , Tumor de Wilms/genética , Tumor de Wilms/patologia , Adulto , Pré-Escolar , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Feminino , Genótipo , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Cariotipagem , Leucócitos Mononucleares/metabolismo , Masculino , Gravidez , Regiões Promotoras Genéticas/genética , Adulto JovemRESUMO
Ependymomas are glial tumours representing approximately 5-10% of all intracranial tumours and are the third most common primary brain tumour in childhood. Only a few karyotypic studies on paediatric ependymomas have been published and no specific chromosomal aberration has been specifically related to this type of cancer. We performed cytogenetic analysis of an ependymoma in an 11-year-old boy. Our patient showed a complex karyotype, characterized by a near-tetraploidy and a sole structural unbalanced aberration: der(2)t(2;11)(q11.2;q13.1), which has not been described before. We here discuss such cytogenetic findings, comparing our data with those reported in the literature.
Assuntos
Neoplasias Encefálicas/genética , Ependimoma/genética , Cariotipagem/métodos , Translocação Genética , Criança , Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Humanos , MasculinoAssuntos
Janus Quinase 2/genética , Fator 88 de Diferenciação Mieloide/genética , Policitemia Vera/complicações , Policitemia Vera/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Substituição de Aminoácidos , Humanos , Imunoglobulina G/metabolismo , Leucina/genética , Masculino , Mutação de Sentido Incorreto , Fenilalanina/genética , Policitemia Vera/patologia , Prolina/genética , Valina/genética , Macroglobulinemia de Waldenstrom/imunologia , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Myeloid neoplasms with eosinophilia and abnormalities of the PDGFRA gene can benefit from therapy with tyrosine kinase inhibitors, therefore revealing the PDGFRA rearrangement is essential to ensure the best choice of treatment. The most common PDGFRA partner is the FIP1L1 gene, generating the oncoprotein FIP1L1/PDGFRA (F/P). In the majority of cases the F/P fusion gene originates from intrachromosomal rearrangement at band 4q12, and occasionally from chromosomal translocations. In both cases, the interstitial chromosomal deletion of a region involving the CHIC2 gene has been reported, which is cryptic by conventional karyotyping but detectable by Fluorescence In Situ Hybridization (FISH) analyses. Herein, we report an acute myeloid leukemia (AML) case presenting with eosinophilia; the F/P fusion gene originated from a new, cryptic and complex intrachromosomal rearrangement of 4q12. Classical FISH assay revealed abnormal hybridization signals, but the presence of the F/P chimaeric gene was demonstrated by molecular analysis. We performed molecular characterization of the chromosomal rearrangement and targeted Next-Generation Sequencing (NGS) analysis with a myeloid gene panel, revealing the presence of pathogenic genomic variants affecting the TET2 and ETV6 genes. These mutations were present as subclones at the disease onset and their clone size increased at relapse.
Assuntos
Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Rearranjo Gênico/genética , Humanos , Cariótipo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , Adulto , Alelos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Feminino , Humanos , Isocitrato Desidrogenase/genética , Masculino , Mutação , Nanoporos , Proteínas Nucleares/genética , Nucleofosmina , Prognóstico , Análise de Sequência de DNA/métodos , Proteína Supressora de Tumor p53/genética , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Chromosomal translocations play a crucial role in tumorigenesis, often resulting in the formation of chimeric genes or in gene deregulation through position effects. T-cell acute lymphoblastic leukemia (T-ALL) is associated with a large number of such rearrangements. We report the ectopic expression of the 3' portion of EST DA926692 in the bone marrow of a childhood T-ALL case showing a t(2;11)(q11.2;p15.1) translocation as the sole chromosome abnormality. The breakpoints, defined at the sequence level, mapped within HPS5 (Hermansky Pudlak syndrome 5) intron 1 at 11p15.1, and DA926692 exon 2 at 2q11.2. The translocation was accompanied by a submicroscopic inversion that brought the two genes into the same transcriptional orientation. No chimeric trancript was detected. Interestingly, Real-Time Quantitative (RQ)-PCR detected, in the patient's bone marrow, expression of a 173 bp product corresponding to the 3' portion of DA926692. Samples from four T-ALL cases with a normal karyotype and normal bone marrow used as controls were negative. It might be speculated that the juxtaposition of this genomic segment to the CpG island located upstream HPS5 activated DA92669 expression. RQ-PCR analysis showed expression positivity in 6 of 23 human tissues examined. Bioinformatic analysis excluded that this small non-coding RNA is a precursor of micro-RNA, although it is conceivable that it has a different, yet unknown, functional role. To the best of our knowledge, this is the first report, in cancer, of the activation of a small non-coding RNA as a result of a chromosomal translocation.
Assuntos
Medula Óssea/metabolismo , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 2/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , RNA não Traduzido/genética , Translocação Genética , Adolescente , Sequência de Bases , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , RNA não Traduzido/metabolismoRESUMO
Two cases of therapy-related acute myeloid leukemia showed complex karyotypes, including a small ring and a larger D-chromosome. Multicolor fluorescence in situ hybridization and bacterial artificial chromosome and fosmid clones showed that both ring chromosomes were composed entirely of material excised from chromosome 12. The deleted segment of 12 was found fused to the short arm of a D-group chromosome. We hypothesized that similar mechanisms were involved in both rearrangements. A fusion at the short arms of chromosome 12 and a D-group chromosome was accompanied by excision and ligation of the chromosome 12 pericentromeric region to form a small ring chromosome.
Assuntos
Cromossomos Humanos Par 12 , Leucemia Mieloide Aguda/genética , Cromossomos em Anel , Cromossomos Artificiais Bacterianos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Mieloide Aguda/etiologia , Masculino , Pessoa de Meia-Idade , Modelos GenéticosRESUMO
We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Nanoporos , Proteínas de Neoplasias/genética , Idoso , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. METHODS: Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. RESULTS: Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. CONCLUSIONS: In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.