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 .

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.

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.

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.

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.

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.

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.

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.

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.

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.