Quantification of the PRAME transcripts in patients with acute myeloid leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the expression level and clinical significance of the preferentially expressed antigen of melanoma (PRAME) transcripts in patients with acute myeloid leukemia (AML).</p><p><b>METHODS</b>Real-time quantitative polymerase chain reaction with EvaGreen dye was established to detect the expression level of PRAME transcripts in the bone marrow mononuclear cells of 56 AML cases and 20 controls. The clinical association of PRAME transcripts was analyzed.</p><p><b>RESULTS</b>The PRAME transcripts were 0-1.46% (median 0.18%) and 0-21 618.09% (median 9.79%) in controls and AML cases, respectively (P< 0.01). Among the FAB subtypes, those with M1, M2, M3 and M4 had significantly higher level of PRAME transcripts than controls. However, those with M5 had similar level of PRAME transcripts as controls. There was a significantly negative correlation between the PRAME transcripts and cytogenetic risk groups (r= -0.438, P= 0.001). Cases in low risk had significantly higher level of PRAME transcripts than those in intermediate and high risk. Among cases with AML-M2, those with t(8;21) had significantly higher level of PRAME transcripts (135.06% -21 618.09%, median 2201.88%) than those without t(8;21)(0.14% -1696.30%, median 17.97%)(P= 0.002). In a patient with sequential samples, PRAME transcripts significantly decreased after induction therapy and significantly increased after relapse.</p><p><b>CONCLUSION</b>The PRAME transcript was highly expressed in AML patients and was a favorable marker of prognosis. Quantification of PRAME transcript can be used in monitoring disease status of AML.</p>

Quantification of GRAF gene expression in patients with acute myeloid leukemia using EvaGreen real time quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To quantify the expression level of GRAF gene in acute myeloid leukemia (AML) and analyze its clinical significance.</p><p><b>METHODS</b>The EvaGreen real-time quantitative polymerase chain reaction (RQ-PCR) assay was established and performed to measure the GRAF gene transcripts in 71 cases with AML and 21 with nonmalignant hematological diseases. The clinical correlation of GRAF expression was analyzed.</p><p><b>RESULTS</b>The established EvaGreen RQ-PCR assay had good specificity, reproducibility and sensitivity. The GRAF expression level was significantly lower in AML (0.01%-169.75%, median 3.82%) than that in controls (14.49%-126.85%, median 56.04%) (P<0.05). There was no correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups (P>0.05).</p><p><b>CONCLUSION</b>The GRAF gene was down-regulated in AML, which might play a role in the leukemogenesis.</p>

Quantification of PML/RAR alpha fusion gene transcripts in patients with acute promyelocytic leukemia by using real-time quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish and evaluate a real time quantitative PCR (RQ-PCR) method for detection and quantification the PML/RAR alpha fusion gene transcripts in patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Three pairs of primers and TaqMan probe were designed for detecting the most frequent PML/RAR alpha transcripts (L-form, S-form and V-form) and normal ABL was used as an internal control. A real time PCR condition was established to detect PML/RAR alpha and ABL positive templates with a series of dilutions. To evaluate this assay, bone marrow samples from 6 APL patients were detected.</p><p><b>RESULTS</b>In repeated tests, maximal sensitivities of 10 copies/microL were obtained, while reproducible maximal sensitivity achieved 100 copies/microL. In 10 normal controls, no amplified fluorescent signals were detected. The median absolute and normalized amount of PML/RAR alpha fusion gene transcripts were 4.27 x 10(3)-3.36 x 10(5) copies/50 ng (median 4.33 x 10(4)copies/50 ng) and 29.38%-600.53% (median 48.12%) respectively. One case showed significant decrease of PML/RAR alpha fusion gene transcripts after induction therapy compared to that at the time of diagnosis, while the fusion transcripts significantly increased after relapsed.</p><p><b>CONCLUSION</b>RQ-PCR is a sensitive, reliable quantitative assay and can be used in the diagnosis of APL and measurement of MRD.</p>

Alteration of methylation status of fragile histidine triad gene promoter in patients with myelodysplastic syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the methylation status of fragile histidine triad (FHIT) gene promoter in patients with myelodysplastic syndrome (MDS) and its clinical relevance.</p><p><b>METHODS</b>Methylation-specific PCR (MSP) was used to detect FHIT promoter methylation in bone marrow samples from 54 MDS cases.</p><p><b>RESULTS</b>Hypermethylation of FHIT promoter was detected in 26 cases (48.1%). Association was not found between FHIT gene hypermethylation and sex, hematologic parameters and chromosomal abnormalities of MDS patients, but found between FHIT gene hypermethylation and age of the MDS cases. Although significant difference was not observed in the frequencies of FHIT gene hypermethylation among patients with refractory anemia/refractory anemia with ringed sideroblasts (RA/RAS) (1/6, 16.7%), refractory anemia/refractory anemia with ringed sideroblasts (RCMD) and refractory cytopenia with multilineage dysplasia with ringed blasts (RCMD-RS) (6/19, 31.6%), refractory anemia with excess blasts-1 (RAEB-1) (7/11, 63.6%), refractory anemia with excess blasts-2 (RAEB-2) (4/7, 57.1%) and refractory anemia with excess blasts in transformation/acute myeloid leukemia (RAEBt/AML) (8/11, 72.7%)(chi-square=8.417, P=0.077), it was observed in patients in early stages (RA/RAS and RCMD) (7/25, 28.0%), advanced stages (RAEB-1 and RAEB-2)(11/18, 61.1%) and RAEBt/AML (8/11, 72.7%) (chi-square=7.938, P=0.019). Furthermore, there was a positive correlation between the frequency of FHIT gene hypermethylation and different IPSS groups (chi-square=10.110, P=0.018).</p><p><b>CONCLUSION</b>FHIT gene hypermethylation might be one of the molecular events involved in the disease progression of MDS.</p>

Expression of PML-RAR alpha fusion transcripts detection by using RQ-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish the real time quantitative PCR (RQ-PCR) assay according to 'Europe Against Cancer' (EAC) program and analyze the results of detection and quantification of different PML-RAR alpha transcript isoforms in patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Three RQ-PCR systems were performed to detect the most frequent PML-RAR alpha transcripts (L-form, S-form and V-form) in 30 APL patients and the RQ-PCR end-point products were identified by electrophoresis.</p><p><b>RESULTS</b>S-form RQ-PCR system could amplify positive signals of three isoforms in all of 30 cases, and V-form RQ-PCR system could do so in both L-form and V-form positive cases, however, L-form RQ-PCR system could only do so in L-form-positive cases. Electrophoresis and sequencing of end-point products amplified by S-form RQ-PCR system revealed three bands in each of L-form (621 bp, 477 bp and 218 bp) and V-form (567 bp, 423 bp and 218 bp) positive patients samples.</p><p><b>CONCLUSIONS</b>RQ-PCR, sensitive and reliable, can be used for monitoring the minimal residual disease in APL patients, however, its results should be interpreted carefully if it is used for detection of PML-RAR alpha fusion transcripts prospectively.</p>