Gene Rearrangement Of LMO2 Gene In Acute T Cell Lymphoblastic Leukemia

Objective: 1. To explore the incidence of LMO2 gene rearrangement in 264 newly diagnosed T-cell acute lymphoblastic leukemia(T-ALL) patients and to detect the m RNA expression of nine oncogenes including LMO2 gene in patients with or without rearrangement, then investigate the relationship of LMO2 gene rearrangement with its expression level. In order to clear the relationship between the LMO2 gene rearrangement, mutations and DNA copy changes, we detected some gene mutations like NOTCH1 gene and arraycomparative genomic hybridization(array-CGH) on part of patients. 2. We seeked the partner genes of 2 T-ALL patients with LMO2 gene rearrangement by the whole genome sequencing(WGS) and tried to clarify the pathogenesis. Methods: 1. 264 cases were studied by R-band karyotypic analysis using short-term culture for chromosomes preparation. Fluorescence in situ hybridization(FISH) and real time fluorescence quantitative polymerase chain reaction(RQ-PCR) were applied on 264 and 49 T-ALL patients for the detection of LMO2 gene rearrangement and nine oncogenes expression including LMO2 gene. Genomic DNA was extracted from frozen bone marrow mononuclear cells(BMMCs) after Ficoll gradient centrifugation using Pure Link Genomic DNA Mini Kit(Invitrogen) according to the standard procedures. NOTCH1 and other genes mutations were analyzed by PCR amplification of the coding region followed by bidirectional DNA sequencing as previous described on 88 T-ALL patients. We detected DNA copy changes by array- comparative genomic hybridization(array-CGH) on 22 T-ALL patients with plenty of DNA samples. Finally, we analyzed synthetically the correlation between LMO2 gene rearrangement, expression, mutation and DNA copy changes in T-ALL. 2. The frozen bone marrow mononuclear cells(BMMCs) of 2 T-ALL patients with LMO2 gene rearrangement were collected and Genomic DNA were extracted using Pure Link Genomic DNA Mini Kit(Invitrogen) according to the standard procedures. Then, we sent more than 4μg DNA to Bo Hao biological technology Co. Ltd. in Shanghai for the whole genome sequencing. We seeked out the partner genes of LMO2 on 2 T-ALL patients by the in-depth analysis of the sequencing results and analyzed the pathogenesis mechanism. Results: 1. The results of LMO2 gene rearrangement, expression, mutation and DNA copy changes in 264 T-ALL patients.A totle of 24 LMO2 gene rearrangement positive patients were foud by FISH in 264 newly diagnosed T-ALL patients(male 21 cases, female 3 cases) and the positive rate is 9.1%. Among the 24 positive patients, karyotypic analysis revealed chromosomal aberrations involving 11p12-13 in 12 patients, comprising 10 with t(11;14)(p13;q11)(10/264,3.8%)and two with del(11p12). Overall, half of LMO2 rearrangements were cryptic by routine karyotypic analysis at a rate of 4.5%(12/264). LMO2 rearrangements were significantly associated with younger age(<18 years), higher hemoglobin concentrations, higher lactate dehydrogenase serum levels and higher frequency of abnormal karyotype(P value <0.05). The were no significant differences between sex, the percentage of bone marrow progenitor cells, white blood cells(WBC) and platelets(PLT) counts(P value >0.05).We screened all 24 LMO2 rearrangement positive T-ALL patients by FISH using BAC clones on methanol/acetic acid-fixed cells obtained from the BM cultures as previously described. For detection of TCRA/D rearrangements, dual color FISH experiments were performed with two contiguous BAC clones: RP11-256C2 and RP11-242H9, respectively labeled with Spectrum Green-d UTPand Spectrum Red-d UTP. We found the karyotype analysis and FISH test were basically identical in 10 patients with t(11;14)(p13;q11) and another 4 cases of T-ALL patients were also detected positive for LMO2 and TCRA/D gene rearrangement which indicate the cryptic t(11;14)(p13;q11). Overall, among the 24 LMO2 gene rearrangement positive patients, karyotypic analysis and FISH revealed chromosomal aberrations t(11;14)(p13;q11) in 14(58.33%)patients, comprising 10 with t(11;14)(p13;q11) by karyotypic analysis and 4 with cryptic LMO2 and TCRA/D gene rearrangement.We found one case of T-ALL patients positive for LMO2 and TCRB gene rearrangement at the same time by the whole genome sequencing. This important finding spurred us to screen TCRB gene rearrangement in the 24 cases with LMO2 generearrangement in T-ALL patients. For detection of TCRB rearrangement, dual color FISH experiments were performed with two contiguous BAC clones: RP11-256C2 and RP11-242H9 and we found 2 cases with TCRB gene rearrangement, including the case with t(7;11)(q35;p13)by WGS.Another case with LMO2 and TCRB gene rearrangements was also positive for TCRA/D gene rearrangement. As the karyotypic result comprising t(8;14)(q24;q11), we hypothesized that TCRA/D gene rearrangement was not result from fusing to LMO2 gene, but with the MYC gene located in 8q24. We confirmed the hypothesis by FISH experiments prepared from BAC clone RP11-440N18. Finally, the TCRA/D gene was fused to MYC gene, while LMO2 gene fused to TCRB gene.LMO2 m RNA expression levels were measured on 10 T-ALL patients with and 39 without LMO2 rearrangements. The RQ-PCR results showed that LMO2 transcripts were significantly higher in cases with LMO2 rearrangements(p=0.02) than without. Meanwhile, the m RNA expression levels of LEF1, LYL1, MEF2 C, STAG2, SEPT1, TLX1, and TLX3 were also measured by RQ-PCR in these patients, showing no differences between patients with and without LMO2 rearrangements with the exception of LEF1. Our findings showed that patients with LMO2 rearrangements had higher LEF1 transcripts(p=0.015) and lower MEF2 C transcripts(P=0.04).To determine the association of LMO2 rearrangements with other recurrent gene mutations in T-ALL, we investigated gene mutations by PCR and direct Sanger sequencing in a cohort of 88 T-ALL patients for whom DNA was available, including 13 with LMO2 rearrangements. Overall, 43 patients(48.9%) had no detected mutations, a single gene was mutated in 25(28.4%) patients, and more than one gene in 20(22.8%) patients. Mutations of FBXW7, IL7 R, NOTCH1, PHF6 and WT1 were respectively present in 8/88(9.1%), 4/88(4.5%), 40/88(45.5%), 12/88(13.7%), and 4/88(4.5%) of patients. FBXW7 mutations were identified in 8/88 T-ALL cases; alone in 2, and associated with NOTCH1 HD mutations in 6 cases. IL7 R mutations were found in 4/88 cases, exclusively affected exon 6; all four cases had an in-frame insertion or substitution at residues P240–S246 of the IL7 R transmembrane domain. NOTCH1 mutations were detected in 40 of the 88 T-ALL patients in the following domains and frequencies: HD, 62.5%; PEST, 25.0%; HD+PEST, 7.5%; and TAD, 5%. PHF6 mutations were identified in 12/88 T-ALL cases; the mutation was missense in two cases, frameshift in four cases, and nonsense in other six cases. In two cases, sites in the same base mutated into the termination codon, which have been reported by others. WT1 mutations were found in 4/88 cases; one patient harboring a missense mutation at codon 462(R462W) located in exon 9, while others all bore frameshift mutations located in exon 7. LMO2 mutations were identified in none of the T-ALL patient samples, while there were two SNPs of the LMO2 gene(rs2038602 and rs3740617). There were no differences in the incidence of mutations of FBXW7(P=0.168), IL7R(P=0.252), NOTCH1(P=0.956), PHF6(P=0.265) and WT1(P=0.252) between patients with and without LMO2 rearrangements.We performed array-CGH analysis on 22 T-ALL samples and found that all patients had one or more genomic abnormalities. A total of 87 genomic alterations were detected, with a mean of 3.9 genomic alterations per sample. Among all of the changes, the highest incidence was deletion of CDKN2A(36.4%, 8/22). Fusion of SET-NUP214, SIL-TAL1, and NUP214-ABL1 were respectively present in 3/22(13.6%), 2/22(9.1%), and 1/22(4.5%) of patients. We identified cryptic copy number changes containing interesting potential candidate genes such as duplication of the MYB(1/22, 4.5%) and deletions of the CDKN2A(8/22, 36.4%), WT1(3/22, 13.6%), LEF1(2/22, 9.1%), LMO2(2/22, 9.1%), together with one each(4.5%) of RB1, PHF6 and ETV6. In 2 patients with microdeletion of upstream of LMO2 gene showed by array-CGH, FISH analysis using a probe from RP11-646J21 and RP11-278N12 confirmed the array-CGH data. 2. The study about the partner genes of 2 T-ALL patients with LMO2 gene rearrangement.We performed whole genome sequencing in 2 LMO2 rearranged T-ALL patients without t(11;14)(p13;q11) or del(11)(p12p13).In case 1(normal karyotype), we identified fusion between 11p13(33,856,828 bp) and 7q34(142,494,025 bp), and we preliminary presumed the chromosomal translocation was t(7;11)(q35;p13). We detected the TCRB gene rearrangement for case 1 by BAC clones RP11-1084E14 and RP11-114L10, and found it was positive.Then, dual color FISH experiments by BAC clones RP11-646J21 and RP11-114L10 confirmed the case was positive for LMO2 and TCRB gene rearrangement at the same time. PCR and bi-directional Sanger sequencing confirmed the presence of chimeric product. The 11p13 breakpoint lay ~23 Kbp downstream of LMO2, while the 7q34 breakpoint lay within the TCRB gene.Thus, we identified a rather rare translocation t(7;11)(q35;p13) in case 1. In case 2, we identified a fusion between 11p13(33,957,035 bp) and 14q32(98,842,615 bp).So we presumed the chromosomal translocation was t(11;14)(p13;q32). The breakpoint of 14q32 lay the downstream of BCL11 B gene. We detected the BCL11 B gene rearrangement for case 2 by BAC clones RP11-2348N10 and RP11-74H1, and found it was positive. Overall, the partner gene of LMO2 in case 2 was BCL11 B gene. PCR and bi-directional Sanger sequencing confirmed the presence of t(11;14)(p13;q32). Conclusions: 1.By the study of the LMO2 gene rearrangement in T-ALL patients, we found the LMO2 gene rearrangement was a recurrent event. The m RNA expression level of T-ALL patients with LMO2 rearrangement were higher than without, and LEF1 gene was interacted with LMO2 gene in T-ALL. There was no correlation between LMO2 gene rearrangement and kinds of gene mutations like NOTCH1 gene, which revealed LMO2 gene rearrangement was the main genetic event in T-ALL patients. 2.We seeked out the partner genes of LMO2 on 2 T-ALL patients by WGS and found the precise positions of the breakpoints. In case 2, we found a novel chromosomal translocation t(11;14)(p13;q32)and novel partner gene of LMO2-BCL11 B gene. We therefore propose that the LMO2 gene should be deregulated by juxtaposition with 3′-BCL11 B via a novel t(11;14)(p13;q32.2) rearrangement.