Objective To establish a rapid method for detection of drug-resistance mutation in HBV, based on PCR-MALDI-TOF MS, and to explore the influential factors on this method. Methods One hundred blood serum samples, which were collected from chronic HBV patients with single drug-resistance or multiple drug-resistance of Lamivudin, Adefovi, Entecavir and Telbivudine, and 10 kinds of mutant HBV plasmids were analyzed using PCR-MALDI-TOF MS and confirmed by PCR-based sequencing. Results Of 100 samples detected, thirty-one samples were positive for drug-resistance and 69 samples were negative. The PCR-MALDI-TOF MS results of 94 samples were completely consistent with PCR-based sequencing. Six samples were inconsistent , of which three samples were positive by the two methods, but more mutation loci were detected by PCR-MALDI-TOF MS than sequencing. The consistent rate of two methods was 94%,detection sensitivity was up to 100 copies/μl, and the cut off value of detectable mutation level was 5%.Conclusion PCR-MALDI-TOF MS could be used for rapid and simple analysis of the drug resistance for the clinical application with features of high sensitivity and accuracy, high throughput and automation.

Objective To compare the curative effect and safety of high-frequency and low-frequency repetitive tran-scranial magnetic stimulation(r-TMS) in the treatment of Parkinson's disease complicated with depressive disorder. Methods All 92 patients with Parkinson's disease complicated with depressive disorder were randomly allocated to three groups. High frequency group received antiparkinsonian drugs combined with high frequency r-TMS (5 Hz);Low frequency group received antiparkinsonian drugs combined with low frequency r-TMS (0.5 Hz);Sham group re-ceived antiparkinsonian drugs combined with sham r-TMS (s-rTMS). Each group received r-TMS twice a week for four weeks. UPDRS and HAMD-17 were assessed every two weeks , adverse events were recorded , and clinical global im-pression was carried out four weeks after the treatment. Results Upon enrollment, scores of HAMD and UPDRS among the three groups were not significantly different (P>0.05). During the second week of the intervention, the HAMD score in high frequency group improved significantly , and the differences between three groups were statistically signif-icant according to one-way ANOVA (P=0.0144). The differences changed larger in the fourth week, with greatly sig-nificant differences between the two groups (P=0.0000);During the second week of the intervention, the differences of UPDRSⅡscores in high frequency, low frequency and sham group were not statistically significant according to one-way ANOVA (P=0.1981). The differences appeared in the fourth week, but the differences between groups were not statistically significant according to one-way ANOVA (P=0.0219). During the second week and the fourth week of the intervention, UPDRS Ⅲ scores in the three groups were not significantly different (P=0.6105, 0.4248 respectively). Adverse events in the three groups were rare, and the differences were not statistically significant. Conclusion High-frequency stimulation on dorsal-lateral left prefrontal cortex is effective in alleviating depression for patients with Parkinson's disease, and is also effective in improving capabilities in daily life, which shows exact clinical effect and rare adverse events.

Objective:To explore the correlation between clinical classification and genotype and prognosis among Chinese children with Very-long chain acyl-CoA dehydrogenase deficiency (VLCADD).Methods:A Chinese pedigree affected with VLCADD admitted at the First People′s Hospital of Yunnan Province in February 2019 was selected as the study subject. The characteristics of disease onset, diagnosis and treatment and prognosis were retrospectively analyzed. Relevant literature was also systematically searched and reviewed.Results:The proband, a 1-year-old boy, had the clinical manifestations of frequently vomiting, hypoglycemia, abnormal liver function and myocardial enzymes. Tandem mass spectrometry screening showed significantly elevated C14, C14: 1, C16: 1, C16: 2, C18 and C14/C8. Genetic testing revealed that he has harbored compound heterozygous variants of the ACADVL gene, namely c. 664G>A (p.G222R) and c. 1345G>A (p.E449K), which were respectively derived from his father and mother. The child was diagnosed with VLCADD cardiomyopathy type and deceased 2 weeks later. Literature review has identified 60 Chinese children with VLCADD. The clinical classifications were mainly cardiomyopathy type and liver disease type, which accounted for 73.3% (43/60). The combination of ACADVL gene variants were correlated with the clinical classifications of VLCAD. Children with one or two loss-of-function (LOF) mutations showed more severe clinical manifestation and a higher mortality. Cardiomyopathy type had the poorest prognosis, with a mortality rate of 76.9% (20/26). C14: 1 may be used as an indicator for the diagnosis of VLCADD, but cannot be used for clinical subtyping and prognosis evaluation. The c. 1349G>A (p.R450H) variant had the highest frequency among the Chinese patients, accounting for 10.8% (13/120). Conclusion:The clinical classifications of VLCADD are strongly correlated with the prognosis, and LOF mutations are more common in those with severe clinical manifestations. c. 1349G>A (p.R450H) may be the most common variant among the Chinese patients, and early screening and diagnosis can greatly improve the prognosis of patients.

Objective:To analyze the genetic mutation characteristics of glucose-6-phosphate dehydrogenase (G6PD) deficiency among infants in Kunming.Methods:A total of 15 533 infants (7 994 males and 7 539 females) born in Kunming from January 1, 2018, to December 31, 2020, with an age range of 2 to 44 days, were selected. G6PD enzyme activity and gene mutation types were detected using fluorescence quantitative analysis, multicolor melting curve analysis (MMCA), and Sanger sequencing. Droplet digital PCR (ddPCR) was used for quantitative analysis of a newly identified variant family to determine the mutant allele proportion in family members. Meanwhile,the protein structure model and pathogenicity prediction of the novel variant were analyzed.Data analysis was conducted using SPSS 26.0. Specifically, chi-square tests were used for the detection rates of G6PD enzyme activity and gene mutations between different genders. One-way analysis of variance (ANOVA) was used for the comparison of enzyme activity among different mutation types.Results:Among 15 533 infants, 143 cases (129 males and 14 females) were tested positive for G6PD activity, with a detection rate of 0.92% (143/15 533). The difference in detection rates between males and females was statistically significant (χ 2=96.76, P<0.001). Out of 89 enzyme activity-positive cases (83 males and 6 females) underwent genetic testing, 77 (72 males and 5 females) were detected by MMCAand other 12 negative samples were underwent further Sanger sequencing, revealing mutations in 6 samples, all of which were males. Among the 83 individuals with gene mutations, 78 had heterozygous mutations, 1 had a homozygous mutation, and 4 had compound heterozygous mutations. A total of 12 mutation types were detected, with G6PD c.487G>A, c.1024C>T, c.1388G>A, and c.1376G>T being the most common, accounting for 74.70% (62/83) of all mutation types. The average G6PD enzyme activity of c.1376G>T was the lowest, and the differences were statistically significant compared to the average enzyme activity of the other three mutations ( P<0.05). One male infant with a newly identified G6PD c.242G>C mutation was detected, predicted to be pathogenic. ddPCR confirmed that the mother of the affected child was a c.242G>C mutant chimera, with a chimera proportion of 6.66%. Conclusions:In the Kunming region, the predominant G6PD deficiency gene mutation is c.487G>A, with the detection of a novel G6PD c.242G>C mutation. The application of ddPCR technology can assist in detecting the proportion of mutation chimeras.

【Objective】 To explore the role and mechanism of TRPC in promoting extracellular matrix (ECM) deposition in rat glomerular mesangial cells (HBZY-1). Methods Immunofluorescence staining was performed to observe the distribution and expression of TRPC1 and TRPC6 in HBZY-1 cells. After AngⅡ stimulation, qRT-PCR and Western blotting were used to detect the mRNA and protein expressions of Gαq/PLCβ4/TRPC signaling pathway main proteins and ECM deposition indicators (α-SMA, collagenⅢ and fibronectin). By silencing the expressions of TRPC1 and TRPC6 by RNA interference, the expressions of ECM deposition indicators were detected. Changes in [Ca²⁺]i influx were determined through Fluo-4AM Ca²⁺ imaging. 【Results】 Both TRPC1 and TRPC6 were expressed in HBZY-1, and were mainly located in cell membrane and cytoplasm. After AngⅡ stimulation, Gαq/PLCβ4/TRPC signaling pathway was activated, and the mRNA and protein expressions of Gαq, PLCβ4, TRPC1 and TRPC6 were all increased (P<0.05). [Ca²⁺]i influx also increased (P<0.01), and the mRNA and protein expressions of ECM deposition indicators (α-SMA, ColⅢ and Fn) were upregulated (P<0.05). Silencing the expressions of TRPC1 and TRPC6 by RNA interference led to decreased [Ca²⁺]i influx (P<0.05), and downregulated mRNA and protein expressions of ECM deposition indicators in HBZY-1 cells (P<0.05). The results suggested that inhibition of TRPC expressions could inhibit AngⅡ induced ECM deposition in HBZY-1 cells, which might be associated with decreased [Ca²⁺]i influx. 【Conclusion】 TRPC may be a novel therapeutic target of renal fibrosis.