Study On The Clinical Characterization, Cytogenetics, FISH And Gene Expressing Profile Of A New And Rare Abnormality Of Chromosome 20-der(20)del(20)(q11q13)idic(20)(p11) In Malignant Hematologic Diseases

ObjectiveA deletion of the long arm of chromosome 20 (del(20q) or 20q-) is the most frequent cytogenetic finding in malignant hematologic diseases with abnormalities of chromosome 20. In malignant hematologic diseases with a solitary chromosomal abnormality, 20q-represents the second most common structural abnormality after the Philadelphia chromosome. But the der(20)del(20)(q11ql3)idic(20)(p11) anomaly had never been reported. We have found this anomaly in 8 cases with myeloid diseases (myelodysplastic syndrome (MDS) 6 cases, acute myeloid leukemia (AML) 2 cases) and 2 cases with acute lymphoblastic leukemia (ALL). In this study, we used cytogenetic, molecular cytogenetic methods and gene chip technique to clarify the entity of this new recurrent cytogenetic abnormality, to determine its clinical and prognostic features, and to explore the genes that may be involved in the pathogenesis and progress of the diseases.MethodsIn part one, we used cytogenetic and molecular cytogenetic methods to identify the entity of this new recurrent cytogenetic abnormality found in 8 cases with myeloid diseases. Chromosome specimens were prepared by direct and/or short term culture of bone marrow cells and karyotype analysis was performed by R- and G-banding technique in all eightpatients. Then fluorescence in situ hybridization (FISH) assay was used in seven of them by five kinds of probes (a subtelomeric probe for 20q, an unique sequence probe for 20ql2, an a-satellite DNA probe for the centromeric region of chromosome 20 and two painting probes for 20p and 20q, respectively ). Meanwhile, clinical and prognostic characterizations of these 8 cases were summarized.In part two, we used the same methods in 2 cases with ALL and ider(20)del(20q). In addition to the karyotype analysis and a panel FISH assay with the same probes used in part one, we used whole chromosome painting probes of chromosomes 9 and 20, a-satellite DNA probes for the centromeric region of chromosomes 9 and 20, and BCR/ABL dual color, dual fusion translocation probe to detect dic(9;20) and BCR/ABL fusion transcript in these two ALL patients.In part three, we ordered a set of BAC/PAC clones which were mapped to 20p to identify the breakpoints of the ider(20)del(20q) anomaly. DNA was isolated and nick translated, and FISH mapping was performed using one probe from a set of BAC/PAC clones along with the centromeric probe for chromosome 20.In part four, we analyed cDNA from 2 cases with MDS by gene expressing profile chip Affymetrix U133A2.0 to screen genes with abnormal expressionResults1. Karyotype analysis showed that one of the normal chromosomes 20 was substituted by one or two small metacentric chromosomes, which were described as ider(20)del(20q), in all eight patients. In seven of them, FISH revealed that all or most metaphases showed 1 ~ 2 small chromosomes which had two symmetric green fluorescent signals (subtelomeric probe for 20q) at both terminals but did not have any red ones (unique sequence probes for 20ql2) between them, two red signals (a-satellite DNA probes for the centromeric region of chromosome 20) in the middle of der(20) and one green signal (painting probes for 20q) which was larger than that of normal chromosome 20, one smallred signal (painting probe for 20p) or yellow signal (combination of painting probes for 20p and 20q) in the middle of the former. The above FISH results demonstrated that following the deletion of 20q, new rearrangement, i.e., idic(20)(pl?l), also took place. Thus, this anomaly should be described as der(20)del(20)(qllql3)idic(20)(pl?l) according to the ISCN(1995). The above eihgt patients with i(20q-) anomaly didn't obtain remission after treatment. At present, seven of them have died with a medium survival of 6.5 months.2. Karyotype analysis showed that one of the normal chromosomes 20 was substituted by one or two small metacentric chromosomes, which were also described as ider(20)del(20q) besides the abnormal karyotypes dic(9;20)(pl3;qll) and t(9;22)(q34;qll), respectively, in these two patients with ALL. The abnormal chromosome 20 was also proved to be der(20)del(20)(qllql3)idic(20)(pll) by a panel FISH assay using the same probes. Meanwhile, FISH assay also validated the existence of dic(9;20) in one patient and t(9;22) in other patient. Of two ALL patients, one had i(20q-) anomaly after relapse of leukemia, and died six month later. The other one have received induction therapy for one month, and has not obtained remission till now.3. The results of D-FISH showed that the breakpoints in 20p were not same in 8 patients with myeloid diseases and 2 patients with ALL. The breakpoints were localized between BAC clone RP11-96L6 and RP13-401N8 corresponding to 20pll.21-20pll.22. At last, the karyotype of i(20q-) anomaly was precisely described as der(20)del(20)(q 11 q 13)idic(20)(p 11).4. The result of gene chip showed the expression of the genes mapped at 20qll.22-20ql3.13 down-regulated, which is consistent with del(20)(qllql2); the expression of genes at 20p no changed, but the expression of most genes at 20q up-regulated; moreover, the expression of FOS gene and most of the TLRs down-regulated.ConclusionsWe discovered a new rare but recurrent chromosome abnormality — der(20)del(20)(qllql3)idic(20)(pll) for the first time in the world, using conventional analysis in combination with FISH assays. It was mainly seen in myeloid diseases including MDS and AML as primary aberration, but was also seen in ALL as secondary aberration. Its appearance may forebode a poor prognosis. Conventienal karyotypic analysis with R banding technique was more useful than G banding technique for identification of i(20q-) anomaly. FISH can revealed its entity in details. The result of gene chip gene revealed preliminarily the change of gene expression profile in patients with i(20q-) anomaly, which may be relavent to the pathogenesis of patients with i(20q-) anomaly. Our study provides important guidance for the correct diagnosis of malignant hematologic diseases with this anomaly and a basis for further investigation of its pathogenesis.