| Objective:1. To comprehensively assess the involvement of ETV6gene mutations in humanhematologic malignancies and then investigated the relationship of gene mutations withrearrangements and its expression level.2. The Array-CGH was utilized to investigate the prevalence and clinical features of12p deletions in60patients with a variety of hematologic malignancie.3. zTo explore the incidence of IgH gene rearrangement in B-cell acute lymphoblasticleukemia (B-ALL) and to analyze its clinical and other cytogenetic features.Methods:1.964cases were studied by R-band karyotypic analysis using direct method and/orshort-term culture for chromosomes preparation. Genomic DNA was extracted from frozenbone marrow mononuclear cells (BMMCs) after Ficoll gradient centrifugation using PureLink Genomic DNA Mini Kit (Invitrogen) according to the standard procedures. ETV6mutations were analyzed by PCR amplification of the entire coding region followed bydirect bidirectional DNA sequencing as previously described. Total RNA was isolatedusing Pure Link RNA Mini Kit (Invitrogen) from bone marrow samples of mutationalcases. After Turbo DNase (Ambion) treatment,1μg total RNA was used for cDNAsynthesis using M-MLV First Strand Kit (Invitrogen),Expression of ETV6was assessedusing the real time quantitative–PCR(RT-PCR).Split-signal FISH was applied on thechromosome samples according to the standard manufacture procedures to analyze theETV6rearrangements.2. Array-based comparative genomic hybridization (array-CGH) analysis was performed in61hematologic malignancies patients for whom at least1.5mg genomicDNA was available. Agilent human CGH microarrays (Agilent Technologies, Santa Clara,CA, USA) containing244000probes (244k) with an average spatial resolution of6.4Kb were used for the array-CGH experiments according to protocols provided by themanufacturer. Image analysis, normalization and annotation were based on FeatureExtraction9.1(Agilent), while data were visualized with Agilent Genomic WorkbenchLite Edition6.5software.3. One hundred and fifty-eight newly diagnosed B-ALL patients were enroled in thisstudy. Conventional R-banding assay was used for karyotypic analysis. Fuorescence in situhybridization (FISH) was performed to analyze the IgH gene rearrangement.Results:1.①A total of15ETV6sequence mutations were detected in15cases, including3inacute myeloid leukemia (AML, n=296,1.01%),3in myelodysplastic syndrome (MDS,n=94,3.2%),2in blast crisis chronic myeloid leukemia (CML-BC, n=69,2.9%),1inchronic phase CML(CML-CP,n=102,1%),3in pre-B cell acute lymphoblastic leukemia(B-ALL, n=139,2.2%),2in chronic lymphoblastic leukemia (CLL, n=49,4.0%) and1inmixed phenotype acute leukemia (MPAL, n=36, n=2.8%). None mutation were found innon-Hodgkin’s lymphoma (NHL, n=36), multiple myeloma (MM, n=28), T-ALL (n=53)and myeloproliferative neoplasms (MPN, n=62). The mutation site analysis showed thatthese mutations affected either the pointed (PNT) domain or the E26transformation-specific (ETS) domain of the etv6. Notably,9out of15mutation wereframeshift mutations and2were nonsense mutations which resulted ETV6translationterminate in advance and predicted to generate a truncated protein. Structural homologymodeling analysis showed that ETV6mutations disorder Tel functions. We investigated theETV6rearrangement in the patients with mutation using split-signal fluorescence in situhybridization (FISH) and found without any translocation existed together with ETV6point mutations. On the other hand, ETV6sequence mutations were often combined withsomatic copy number deletions, therefore the amplification and deletion were evaluated using realtime-PCR, while the result showed that the ETV6without expression changes(p=0.7), amplification and deletion.②FISH was used to determinated the rearrangement of ETV6in hemotologicmalignancies harboring karyotypic abnormalities at chromosome12. Of the73casesdetected by FISH, rearrangement was detected in7case(9.6%),haploid deletion wasobserved in12patients(16.4%), partial deletion was observed in6patients (8.2%).Of the7rearrangement positive patients,including4in AML(4/24,16.7%),2inALL(2/11,18.2%),1in CML-CP (1/5,20%) and3in male(3/44,6.8%),4infemale(4/29,13.8%). Of which3chromosomal translocations were t(12;22)(p13;q12),t(3;12)(q26;p13) and t(12;17)(p13;q21),they have already been reported before,but in therest abnormalities such as t(12;20)(p13;q11),t(12;14)(p13;q21),del(12)(p12),t(12;13) thepartners of ETV6gene is still unknown.2.12p deletions were identified in19.6%(12/61) hematologic malignancies patientsanalyzed. Among these,2in AML(n=9,22.2%),6in ALL (n=22,27.2%),2in MPAL(n=15,13.3%),1in each of CML and NK cell leukemia.But none were found in MDS andMPN. The deletion regional focus in12P12.3-12P13.2(11.4MB-17.0MB), and11of thesepositive patients also accompany with ETV6gene deletion(91.7%).Of the12patients with12P deletions,the median Hb was62.8g/L(38-100)g/L,and Hb counts were lower for12pdeletion patients compared with non-deletion patients(P<0.05). But the Wbccounts,Platelet counts,Sex ratio,survival time of patients with deletion patients andnon-deletion patients were similar.3.IgH gene rearrangements were found in6.9%(11/158) patients, and the percentage ofIgH gene rearrangement positive cell ranged from10.5%to88.2%. Among11B-ALL caseswith IgH rearrangements showed by FISH analysis,2cases presented normal karyotype. In9cases with chromosomal abnormalities, complex karyotype was detected in8cases andrearrangements involving14q32were found in5cases. The incidence of IgH generearrangement in those cases with complex chormosome abnormalities were higher than thatin normal karyotype and non-complex chromosome abnormalities (8/55vs2/52vs1/51, P<0.05).Conclusions:1.By this systematic study of the ETV6gene mutation,we find that ETV6genemutation is a recurrent event, can be seen in MDS,B-ALL, T-ALL, CML, CLL and otherhematological malignancies. A variety new types of mutations have been found. Thesemutants have a significant impact on the function of ETV6.2.In this study,Array-CGH technology be used for detecting the incidence of12Pdeletion in hematologic malignancie.Identify the presence of12P deletion as a recurrentgenetic alterations,verify the common deletion region in12P12.3-12P13.2(11.4MB-17.0MB).And find that most of the12P deletions accompanied with the alterations ofETV6gene.3.The IgH gene rearrangement is a recurrent cytogenetic abnormality in B-ALL. FISHanalysis plays an important role in identifying the rearrangements involving the IgH gene. |
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