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.