Mutational profile of myeloproliferative neoplasms detected by a customized NGS-based gene panel / 中华检验医学杂志

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.

Performance evaluation of the automated SNPs analyzer for the detection of JAK2 V617F mutation / 中华检验医学杂志

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.

Research progress and application of nocoding RNAs in Cancer / 中国生化药物杂志

With the rapid development of modern life sciences, tens of thousands of noncoding RNAs have been discovered, their biological roles have been revealed.Non-coding RNA, as a research hotspot in the field of molecular biology, has been shown to regulate the development and progression of tumors.This paper mainly describes the current research of several non-coding RNA(miRNA, lncRNA, circRNA, tRF)in the regulation of tumor and its application in the precision medicine era.

High-resolution melting analysis:applications in molecular tumor testing / 中华检验医学杂志

High-resolution melting technique shows great potential as a clinical diagnostic tool because of its cost-effectiveness , convenience and closed-tube format which can prevent the risk of cross contamination.HRM analysis has been widely used in molecular diagnostics in the management of cancer by mutation detection , pre-sequence screening , methylation and copy number variations , which involves cancer screening, diagnosis, individual treatment and prognosis .Take advantage of the high detection sensitivity of HRM, the accuracy of mutation screening method could be improved and the test cost could be reduced by combing HRM analysis with sequencing , which helps to develop the molecular diagnostic technique .

Detecting JAK2 V617F mutation in myeloproliferative neoplasms by microarray based digital PCR / 中华检验医学杂志

Objective To evaluate the sensitivity, repeatability and accuracy of microarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms (MPN).Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera(PVs),11 primary thrombocythemias (ETs) and 2 primary myelofibrosis (PMFs), were collected from Huashan Hospital, Fudan University during 2014 -2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved.Human erythroleukemia cell line ( HEL ) and colorectal cancer cell SW480 were used as the mutant and the wild type control, respectively.The sensitivity of microarray digital PCR were verified by detecting the gradient diluted mutation standard harboring 30%, 10%, 1%, 0.1%and 0.01%mutant allele burden, respectively .Repeatability was evaluated by detecting 1%and 10% mutated samples for 5 times, respectively.MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR.Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1%, which is approximate to 0.16 copies per microliter.The results obtained from the two kinds of technique showed a high correlation by linear regression analysis (R2 =0.998 3).The results of repeated samples showed CVs as 17.18% for 1%mutant allele burden and 7.50%for 10%.Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR.In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR.Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.

Simultaneous detection of JAK2 V617F mutation and JAK2 exon12 mutations by multiplex PCR and high-resolution melting curve analysis / 中华检验医学杂志

Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.

Study on the relationship between HLA-B * 5801 and severe cutaneous adverse reactions caused by allopurinol / 中华风湿病学杂志

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.

Establishment and analytical validation of JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis / 中华检验医学杂志

Objective According to the requirements of the College of American Pathologists (CAP),to validate the laboratory developed test-JAK2 V617F mutation.Methods This is a clinical laboratory diagnosis study.Fifty-seven peripheral blood and bone marrow samples were collected from the BCR/ABL-negative myeloproliferative neoplasms (MPN) patients in Huashan Hospital from Apr 2010 to Dec 2010.JAK2 V617F mutation was detected by fluorescence PCR combined with melting curve analysis.The accuracy,precision,analytical sensitivity,and anti-interference ability of the assay were validated.The Kappa test was used to evaluate accuracy.Coincidence rate was used to evaluate the repeatability.Paired t test was used to evaluate analytical sensitivity.Results There was a close agreement between the reference method(sequencing) and the test method (fluorescence melting curve analysis) (Kappa =0.89).The coincidence rate was 100％.The results of the assay was not affected by lipoprotein (＜ 24 mmol/L) or bilirubin (＜450 μmol/L).The detection limit of the assay was 5％.Conclusion JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis could be applied to the clinical laboratory.

Screening MPL515 mutations from 286 cases of essential thrombocythemia and primary myelofibrosis / 中华检验医学杂志

ObjectiveTo establish a simple and sensitive method to detect MPL515 mutations in peripheral blood of ET and PMF patients,and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients.MethodsTotallv 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases.SYBR GreenⅠreal-time PCR was used to detect JAK2V617F mutation.Taqman probe was designed to be specific for the three types of mutations ( MPl515wt,MPLW515L and MPIW515K).Real-time PCR was used to detect MPL515 mutations.Tbe results were confirmed by sequencing after T-A cloning.Results Among 261 ET patients,119 cases (45.6％ ) were identified as JAK2V617F mutation carriers and 7 cases (2.7％ ) were detected to be MPl515 mutation carriers,including 5 cases with MPLW515L,1 case with MPLW515K and 1 ease with MPLW515L + K.Additionally 10 cases with JAK2V617F(40.0％ ) and 3 cases with MPL515 ( 12.0％ ) were screened out in 25 PMF patients,including 1 case with MPLW515L and 2 cases with MPLW515L + K.One ET patient was found to harbor concurrent JAK2V617F and MPL515 mutations.ConclusionJAK2V617F mutation is the major molecular marker of ET and PMF,meanwhile MPL515 mutation is important and useful complement.

The association between JAK2 46/1 haplotype and the susceptibility of PV and ET in Chinese Han population / 中华检验医学杂志

Objective To investigate the frequency of JAK2 46/1 haplotype,tagged by the C-allele of SNP rs 12343867(C/T) in Chinese Han patients with PV and ET,and study the relationship between the JAK2 V617F mutation.Methods The whole blood was collected from 125 PV patients,87 ET patients and 213 healthy people.The JAK2 46/1-linked tagged SNP was screened and genotyped with high-resolution DNA melting analysis.20 random selection copies was certificated by DNA sequencing.The frequencies of genotypes and alleles at tSNP was compared between the case and healthy groups by the chi-square test.Results The C-allele frequency of 125 PV patients was 62.8% and T-allele was 37.2%.Also C-allele frequency of 87 ET patients was 45.4% and T-allele was 54.6%.The random selection copies was verified by DNA sequencing.The results showed that the distribution of JAK2 46/1 tSNP genotypes were significantly different(x2 =78.69,P＜0.01).CC and CT had a higher risk for MPNs compared to TT homozygotes(CC vs TT OR = 18.56,95 % CI = 8.70-39.58; CT vs TT OR = 3.60,95 % CI = 2.28-5.69,all of P＜ 0.01).SNP rs 12343867 was genotyped in 212 patients with concomitant analysis of V617F allele burden.Cenotype distributions did not show significant difference compared with JAK2V617F in PV patients(x2 = 2.47,P =0.12).But in ET patients,compared with V617F-negative,the frequency of C-allele showed difference (x2 =7.75,P＜0.01).Conclusions The incidence of the JAK2 46/1-linked C allele is significantly increased the disease risk of PV and ET.Results indicate that JAK2 46/1 haplotype is associated with an increased risk of acquiring a specific somatic mutation.

Establishment of HRM method for quantitative determination for methylation level of DLC-1 promoter in prostate cancer and its clinical significance / 中华检验医学杂志

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.

The determination of a new single nucleotid polymorphism intron 5 (+4668) with high-resolution melting technology in gout patients / 中华风湿病学杂志

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.

Screening the factors which affect α-toxin expression of CA-MRSA / 中华检验医学杂志

Objective To screen the factors that can affect α-toxin expression of CA-MRSA except for quorum-sensing system and to investigate the regulative mechanism of the interesting genes. Methods S. aureus CA-MRSA transposon mutagenesis library was constructed by using mariner based transposon mutagenesis system. The clones with significantly changed level of hemolysis were selected, the location of erm insertion in a gene was confirmed by arbitrary primed (inverse) PCR and nucleotide sequence. Genetic complementation, mice bacteremia and skin abscess models and real time RT-PCR were used to study the function of the interesting gene. Results Twenty-five mutants with down-expression of α-toxin were selected by screening about 104 isolates of transposon mutagenesis library. The hemolytic diameter of CA-MRSA wild type was about 212 mm, no clear hemolysis was found in AraC-, The hemolytic diameter of AraC-pT181 araC was about 197 mm. Real time RT-PCR results showed that compared to the expression of the virulence factors in CA-MRSA wild type( PSMα 257. 30 ±37. 33 ;agr 115. 60 ±0. 81 and α-toxin 3.23 ±0. 21), in AraC-, α-toxin, PSMα and agr were significantly down regulated(α-toxin 1.09 ±0.01 :t = 10. 18, P <0.01 ;PSMα 34.85 ±2. 15:t=5.95,P<0.05;agr35. 19 ±1. 72:t =42. 33, P<0. 01). The result of mice bacteremia model showed that the virulence of wild type and AraC- ( (x) ± s ) were significantly different (x2 = 21. 34, P < 0.01). The expression of PSMα, agr and α-toxin in AraC-pT181araC ( PSMa 180.10 ± 15.29;agr 101. 50 ±8. 96;α-toxin 2.59 ±0.26) had no significant difference compared to the expression of the virulent factors in CA-MRSA wild type (PSMα: t =1.914, P>0.05;agr:t= 1.563, P>0.05;α-toxm: t = 1. 923, P > 0. 05 ). There were no significant difference of the expression of ClpP in AraC-(0. 21 ±0.01) and in AraC-pT181araC(0.17 ±0.03)compared to the expression of ClpP in CA-MRSA wild type (0. 20 ± 0.01) (t=0.555, P>0.05 and t=0. 851, P>0.05). The result of mice skin abscess model showed that the dermonecrosis area caused by CA-MRSA was (136. 5 ±21.45) mm2, the dermonecrosis area caused by AraC- was (55. 69 ± 13. 81) mm2, the different was significant (t = 3.169, P < 0. 05). Conclusion In CA-MRSA, AraC-type transcriptional regulator controlled the pathogenesis of CA-MRSA by regulating the expression of the most important virulence factors such as hla, PSMα and agr.

Detection of JAK2 V617F mutation by real-time PCR / 中华检验医学杂志

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.

Association of HLA-DRB1*03,*04 and*11 alleles with alopecia areata in Han Nationality in East China / 中华皮肤科杂志

Objective To investigate the association of HLA-DRB31*03,*04 and *11 alleles with alopecia areata(AA)in Han Nationality in East China.Methods Polymerase chain reaction-sequence specific primer(PCR-SSP)method was conducted in 158 Chinese Han patients with AA as well as in 172 healthy human controls in East China.The relationships of HLA-DRB1 polymorphism to age of onset,episode frequency,clinical course,family history,and severity of AA were evaluated.Results No significant differences were observed for the frequency of HLA DRB1*03,*11 alleles between the patients and human controls,while increased frequency of HLA-DRB1*04 was observed in patients(OR=1.99,Pc=0.01).Multiple logistic regression analysis revealed that HLA-DRB1*04 was more prevalent in patients with an onset after 16 years of age(OR=1.94,Pc=0.02),those without family history(OR=1.97,Pc=0.02),those with recurrent AA(OR=2.49,Pc=0.02),those with a clinical course of more than 1 year(OR=2.94,Pc=0.01),those with severe AA(OR=3.53,Pc=0.00)and tbose with single episode of AA(OR=1.83,Pc=0.04)in comparison with the normal human controls.Conclusion This study demonstrates that HLA-DRB1*04 allele is associated with the occurrence and clinical types of AA in Han Nationality in East China.