ABSTRACT
Objective By a sequencing panel consisting of 50 targeted genes, aiming at depicting the molecular landscape of ET, PV, and PMF, which are three major subtypes of MPN, to provide valuable information in the diagnosis and prognosis of MPN.Methods A retrospective study was conducted of 53 patients from Huashan hospital and Changhai hospital. All patients were diagnosed in accordance with the 2016 WHO diagnostic criteria for MPN, including 31 cases of ET(11 males, 20 females, median age 55 years), 17 cases of PV(12 males, 5 females, median age 65 years), and 5 cases of PMF(4 males, 1 females, median age 67 years), and underwent next-generation of DNA sequencing of their bone marrow or blood samples. The genetic analyses were performed on bone marrow or peripheral blood. Referring to COSMIC, dbSNP, Clinvar and other public databases, we analyzed the sequencing data, and elucidated the mutation profile of MPN patients, combining with their clinic information. Results In addition to the typical JAK2, CALR, and MPL mutations, pathogenic mutations in other 11 genes were detected, as well as 4 SNPs that confer individual susceptibility to MPNs (rs4858647, rs9376092, rs58270997, rs621940). The average rate of mutated genes was 2.3 genes per patient. In all patients (53 cases), the mutated genes detected were TET2, EZH2, ASXL1, MIR662, SF3B1, BARD1, DNMT3A, KIT, RUNX1, TP53, NRAS according to their mutational frequency. Conclusions Applying next-generation sequencing technology, multi-gene sequencing of a bunch of typical BCR-ABL-negative MPN patients can be performed at one time within 2 working days, and pathogenic mutations other than JAK2, CALR, MPL can be found, which has a bright prospection in clinic.
ABSTRACT
Objective To test and evaluate the JAK2 gene V617F mutation in patients with myeloproliferative tumors based on i-densy IS-5320 platform according to ISO15189 accreditation requirements.Methods Instrument performance verification.Selected from December 2014 to February 2017, 20 cases of JAK2 V617F mutation positive peripheral blood samples from Huashan Hospital of the Shanghai FuDan University Medical College and 20 cases of peripheral blood samples with negative JAK2V617F mutation.The Realtime PCR with TaqMan MGB probe was selected as the control method to verify and evaluate the accuracy of testing JAK 2 V617F mutation on i-densy IS-5320 platform.Whole blood samples were used to evaluate the reproducibility , cross-contamination and anti-interference ability of this platform.The ability of mutation load was verified by detecting mixtures of human erythnoleukemia cells and colorectal cancer cell HCT116 with 12 different proportions.Results I-densy IS-5320 platform and TaqMan MGB probe real-time fluorescence quantitative PCR show the same result .The within-run reproducibility and between-run reproducibility are both 100%.There is no observed contamination .High bilirubin and high triglyceride blood samples have no obvious interference on mutation detection .The mutation ratio with a load as low as 0.25%could be tested stably by i-densy IS-5320 platform.The detecting peak of melting curve can reflect the ratio of JAK2 V617F mutation to some extent.Conclusions I-densy IS-5320 can detect the mutation of JAK2 V617F gene in the whole blood directly.It has high sensitivity, accuracy and stability, and is easy to operate, and also can reflect the mutation load of JAK2 V617F, which could meet the clinical requirements for the detection of mutations.
ABSTRACT
Objective To investigate the relationship between HLA-B * 5801 allele and severe cutaneous adverse reactions caused by allopurinol or other drugs.The clinical value of HLA-B * 5801 as the marker of allopurinol-SCAR was evaluated.Methods Forty-three patients with allopurinol-SCAR,133 patients without SCAR after taking allopurinol for 3 months were included.Ninety-six patients with SCAR caused by other drugs and 148 healthy individuals were enrolled into the present study.HLA-B * 5801 allele was detected by PCR-SSP method.Data were analyzed by chi-square test.Results HLA-B * 5801 was present in 40 of 43 (93.0％) patients with allopurinol-SCAR,which was significantly higher than 19 of 148 (12.8％) in healthy subjects (x2=100.353,P＜0.01,OR=90.5,95％CI 25.5-321.8).But there were no significant differences between allopurinol-tolerant patients and healthy controls(10 of 133,7.5,x2=2.141,P＞0.05,OR=0.6,95％CI 0.2-1.2).And there were only 14 of 96 (7.5％) patients with SCAR caused by other drugs had HLA-B * 5801 (x2=0.152,P＞0.05,OR=1.2,95％CI 0.6-2.4).Conclusion The study indicates that people with HLA-B * 5801 have a high risk of allopurinol-SCAR.HLA-B * 5801 is a specific and predictive marker for guiding the selection of uric acid lowing drug allopurinol.
ABSTRACT
Objective To establish quantitative method for detection of methylation level of DLC-1 promoter with HRM technology to analyze its association with pathological parameters in prostate cancer.Methods 89 prostate cancer tissue samples and 10 matched normal tissue samples were enrolled into this study.Prostate cancer cells were obtained by LCM.DNA was extracted and modified for methylation determination.CpGenome Universal Methylated DNA was chosen as 100% methylation sample.Then the calibrators representative of 100%,80%,50%,30%,10% and 0% methylation levels were prepared with dilution in a DNA sample of peripheral blood from healthy subjects(100% non-methylation).The DLC-1 methylation levels in prostate cancer tissue samples were detected with HRM.The associations of methylated level with age of patients,PSA value,TNM stage were investigated respectively.ResultsThe melting curves representing 100%,80%,50%,30%,10% and 0% methylation levels were aligned from right to left.The methylation levels of 10 adjacent normal samples and 35 prostate cancer samples were overlapped with 0% methylated calibrator.The methylation levels of 5 cancer samples ranged between 0% and 30%.The methylation levels of 29 cancer samples ranged between 31% and 80%.The methylation levels of 20 cancer samples ranged between 81% and 100%.HRM could be used to reliably detect the as low as 10% methylation for each assay,whereas methylation specific PCR(MSP) could be used to detect 30% methylation level.No significant association between methylation level and patients' age(X~2=3.29,P=0.19),PSA level(X~2=2.04,P=0.36) was found.However,DLC-1 methylation was higher in the prostate cancer tissues with advanced TNM stage(X~2=9.04,P=.01).Conclusions The quantitative method for DLC-1 methylation with HRM is successfully established.It is convenient with good reproducibility and high sensitivity.DLC-1 methylation could be used as the molecular marker for estimation of malignancy in prostate cancer.
ABSTRACT
Objective To investigate a new single nucleotid polymorphism (SNP) intron5(+4668C/T) in SLC22A12 in primary gout patients and the association between clinical characteristics and genotypes. Methods One hundred and one primary gout patients and 186 healthy subjects were recruited into this study. Blood pressure, body mass index (BMI) was recorded. Serum uric acid, glucose, lipid and creatinine were detected. DNA was extracted from peripheral blood to amplify the fragment located in intron 5. The genotypes of SLC22A12 can be detected with high-resolution melting (HRM) assay, followed by sequencing analysis. Chi-square test was used for statistical analysis. Results ① A new SNP in intron 5 of SLC22A12 was identi-fied successfully by HRM, which was defined as intron 5 (+4668C/T). CC, CT and TT genotypes were unam-biguously distinguished with HRM technology, which was fully concordant with sequencing. ②The genotypes of CC, CT and TT in male and female groups were 28.1%, 33.7%, 38.2% and 20.0%, 47.1%, 32.9%, respectively.③ However, no significant differences of genotype distribution were found concerning BMI, blood pressure, creatinine, total cholesterol and triglyceride in both male group and female group. But the serum uric acid levels in the CC genotype were significantly higher than those with the CT+TT genotypes. ④ The genotype frequencies of CC and CT+TT in high uric acid group were remarkably different from those in low uric acid group (21.2%, 78.8%,; 35.0%, 65.0%; P<0.05). Conclusion A new SNP has been successfully discovered with HRM technology with simplicity, rapidity and accuracy. T allele of intron 5 (+4668C/T) may be a genetic protective factor for hyperuricemia among Chinese population.
ABSTRACT
Objective To establish real-time PCR method for the detection of JAK2 V617F mutation and evaluate its clinical significance in patients with myeloprollferative disorders and leukemia.Methods 71 chronic myelocytic leukemia(CML) patients, 22 essential thrombocythemia (ET) patients, 11 primary myelofibrosis (PMF) patients, 9 polycythemia vera (PV) patients and 7 cosinophilia patients were enrolled in this study. JAK2 V617F mutation was determined by real-time PCR and amplification refractory mutation system (ARMS), followed by sequencing. Human erythroleukemia cell (HEL cell)DNA was used as homozygous control of JAK2 V617F mutation. The detection limit for either real-time PCR or ARMS was evaluated. Results Real-time PCR assay showed that there was a melting temperature(Tin) peak at (75.0±0.2)℃ for wild type samples and a Tm peak at (76.6±0.2)℃ for mutation type samples. JAK2 V617F mutation was detected in 8(88.9%) patients with PV, 12(54.5%) patients with ET and 7(63.6%) patients with PMF respectively. But there was only one positive case in 71 CML patient (1.4%). The results showed complete concordance with ARMS results and confirmed by sequencing. The mutation could be detected in 102 HEL cells per 106 white blood cells by real-time PCR, whereas the mutation can be assessed in 104 HEL cells per 106 white blood cells by ARMS. Thus, the sensitivity of real-time PCR was 100-fold higher than ARMS. Conclusions The real-time PCR method is successfully established for detection of JAK2 V617F mutation. This method is more sensitive, convenient than ARMS, and suitable for clinical application. There is high frequency of JAK2 V617F mutation in myeloproliferative disorders and it could be used as the diagnostic marker for myeloproliferative disorders.