Objective To develop a convenient, rapid and specific method using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) for detection of facioscapulohumeral muscular dystrophy(FSHD). Methods Genomic DNA was extracted and digested by restricted endonuclease EcoR Ⅰ , followed by agarose electrophoresis. The DNA (< 38 kb) was retrieved from agarose electrophoretic gels. The primers and probe were designed in D4ZA gene in chromosome 4. One hundred and fifteen subjects were examined by FQ-PCR using the retrieved DNA (<38 kb) as a template and the result was analyzed by fluorescent curve comparing with positive control. Results The results by FQ-PCR showed that 13 cases were positive in 16 FSHD cases whose EcoR Ⅰ fragment sizes were known, 75 cases were negative in 78 cases of normal controls, 15 cases were positive in 16 FSHD cases diagnosed clinically whose EcoR Ⅰ fragment sizes were unknown, and 3 cases were positive in 5 cases of relatives of FSHD patients. Consistency was checked using Kappa index between the 2 gene diagnostic tests for FSHD (FQ-PCR test and the traditional Southern blotting test), and between the 2 diagnostic criterions (gene diagnosis by FQ-PCR and clinical diagnosis). The results were statistically significant (κ = 0. 765, P = 0. 002 ; κ = 0. 844, P = 0. 000). Conclusions A new genetic diagnostic method of FSHD by FQ-PCR was developed, which was more simplified and reliable compared to the time-consuming, radioactive Southern blotting. It could also detect the D4Z4 arrays in cases having deletion of p13E-11 as well as the interchromosomal exchange between 4q35 and 10q26. The new method of FQ-PCR for FSHD may be extended to utilize clinically in future.

Objective To investigate the features of lipid ratios in patients with newly diagnosed T2DM, and the effects of intensive insulin treatment on them. Methods 90 patients with newly diagnosed T2DM and 58 matched people with normal glucose were enrolled to assess height,weight,waist circumference,blood glucose and lipid profiles. BMI,TC/HDL-C,TG/HDL-C,log(TG/HDL-C),LDL-C/HDL-C,HOMA-B and HOMA-IR were calculated respectively. All the patients received the continuous subcutaneous insulin infusion with insulin pump. The treatment continued for more 10~14 days after blood glucose reached the standard. All the above indi-cators were reexamined after treatment. Results Dyslipidemia in patients with newly diagnosed T2DM mainly showed as hypertriglyceridemia and decreased HDL-C compared to the control group(P<0.05). TC/HDL-C,TG/HDL-C,log(TG/HDL-C)and LDL-C/HDL-C significantly increased in these patients(P<0.01). After short-term intensive insulin therapy,all lipid ratios were significantly decreased and the changes of lipid ratios were positively correlated with the change of HOMA-IR(P<0.05). Conclusion Short-term intensive insulin therapy for patients with newly diagnosed type 2 diabetes can significantly lower the lipid ratios related to HDL-C. The effects may be closely related to improvement of insulin resistance.

Objective:To investigate the clinical features of patients with Langerhans cell histiocytosis (LCH), and analyze the association between BRAF V600E mutation status and clinical features. Methods:A retrospective analysis was carried out for the clinical data of 60 patients with LCH at the Department of Pediatric Oncology, Sun Yat-sen Memorial Hospital between April 2013 and December 2019.Among them, 39 patients undertook BRAF V600E mutation testing, which in paraffin-embedded tissue samples were detected by quantitative real-time PCR (qRT-PCR), and in peripheral blood and/or bone marrow were tested by high-throughput sequencing, for analyzing the correlation between BRAF V600E mutation and clinical characteristics of LCH. Results:(1)Clinical characteristics: the age of 60 LCH patients was (4.08±0.45) years, with 43 male cases and 17 female cases.Patients at young age (≤2 years) and with risk organ (RO+ ) and central nervous system (CNS) risk lesions involvement were concentrated in the multisystem involvement (MS) group ( P<0.05). (2)Therapeutic response after induction therapy: the response to induction therapy was achieved in 28 of 60 treated patients (41.7%) and 32 (53.3%) did not.After excluding stratification factors of treatment regimen, MS ( OR=6.855, 95% CI: 2.077-22.622, P=0.002) and the age≤2 years ( OR=4.944; 95% CI: 1.601-15.275; P=0.005) were risk factors in poor chemotherapy response.RO+ ( OR=8.250, 95% CI: 1.617-42.090, P=0.005) was a significant risk factor for a poor chemotherapy response in JLSG-02 treatment group.Differently, RO+ had no dramatic effect on chemotherapy response in CCHG-LCH-2019 treatment group.(3) BRAF V600E mutation: 39 patients were determined BRAF V600E status, with the positive rate of BRAF V600E mutation in paraffin-embedded tissue samples reaching 70.3%(26 cases). BRAF V600E mutation was not associated with early treatment response, age, sex, MS and RO+ ( P>0.05). However, the positive rate of BRAF V600E in children with MS and CNS risk lesions was higher than the controls, with 76.0% (19 cases) vs.57.1% (8 cases) and 74.1% (20 cases) vs.58.3% (7 cases), respectively.Totally, 3 of 8 cases were positive in bone marrow, with 2 cases of MS, and 1 case of multiple bone invasions, and 1 of 5 cases was positive in peripheral blood, with liver and spleen being involved. Conclusions:LCH patients with age≤2 years, MS and RO+ exhibited a poor response to initial treatment, required for more aggressive treatment strategy.Lesion with activating BRAF V600E mutations suggests that LCH is a clonal disorder.There may be great variability between BRAF V600E mutations and MS as well as CNS risk lesions.In the mutation dataset, part of patients had positive BRAF V600E mutations in bone marrow/peripheral blood.This might suggest a different pathogenesis in such patients, has a certain clinical sense in some aspect.