[Analysis of clinical phenotype and genotype of Chinese children with disorders of sex development].

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

[The establishment and multicenter application of internal quality control system for real-time quantitative PCR detection of BCR-ABL (P210) transcript level].

Objective: The study aims to establish a perfect BCR-ABL (P210) internal quality control system and ensure the long-term stability and comparability of the detection results between laboratories and to popularize and apply it in the three hospitals. Methods: The Qilu Hospital of Shandong University (H1) prepared a set of the BCR-ABL (P210) quality control substances to establish and improve internal quality control system. We went to other three participating hospitals (H2, H3, and H4) to inspect quality control before the measurement. In addition, we mailed 25 sets of quality control substances to each of the hospital for detection. The slope and intercept of the standard curve of each hospital and the detection results were analyzed and statistically judged using the Levey-Jennings quality control chart combined with the Westgard multirule theory. Then, we made a quality control evaluation. Results: ①An internal quality control system for the BCR-ABL (P210) transcript levels monitoring was successfully established for the quality inspection before the measurement, statistical judgment during the measurement, and evaluation after the measurement. ② Both the slope and intercept of the standard curve of the four hospitals was under control. ③The multicenter quality control substance judgment results were as follows: for H1 hospital, two times of "1(2s)" warning were found in the middle-level quality control substance, which was judged as being under control; for H2 hospital, one time of "1(2s)" warning was found for each quality control substance, which was judged as being "2(2s)" out of control; for H3 hospital, its high-level quality control substance violated the "1(3s)" rule, and low-level quality control substance appeared "1(2s)" warning, which was judged as "1(3s)" out of control; and all quality control substances were under control in H4 hospital. ④The quality control evaluation and correction were as follows: two hospitals were under control, and the other two hospitals had an "out of control." We found out the reason for the out of control and corrected them. ⑤The comparisons of the original values of the multicenter quality control substance were as follows: there were statistical differences in the results of high-level quality control substance among the four hospitals, and no significant difference was found in the results of the medium-level and low-level quality control substance. ⑥The comparisons of the IS values of the multicenter quality control substance were as follows: the IS values of the three quality control substance in H2 and H3 hospitals were significantly higher than those of H1 hospital, and H2 hospital was significantly higher than H3 hospital. Conclusion: A perfect and stable internal quality control system for the BCR-ABL (P210) transcripts has been established, which can effectively ensure the accuracy and stability of the clinical detection results. This internal quality control system has been successfully popularized and applied in other hospitals.

[Clinical features and survival analysis in non-M(3) acute myeloid leukemia patients with ASXL1 gene mutation].

Objective: To examine the survival rates and clinical characteristics of people with newly discovered non-M(3) acute myeloid leukemia (AML) who carry the ASXL1 gene mutation. Methods: From January 2016 to April 2021, the clinical information of patients with newly diagnosed non-M(3) AML at Shandong University's Qilu Hospital was retrospectively examined, and their clinical characteristics and survival were compared and analyzed. Gene mutation was detected by next-generation sequencing. Results: ① The study included 256 AML patients who were initially diagnosed and had complete data, including 47 cases of ASXL1 gene mutation-positive (ASXL1(+)) patients and 209 cases of ASXL1 gene mutation-negative (ASXL1(-)) patients. All patients were divided into three groups: elderly (≥60 years old, n=92) , middle-aged (45-59 years old, n=92) , and young (≤44 years old, n=72) . ②WBC, and age were higher in patients with ASXL1 mutations compared to ASXL1(-) patients, while complete response after the first round of treatment (CR(1)) was lower (P<0.05) . In the elderly group, WBC and the proportion of aberrant cells in nuclear cells in ASXL1(+) patients were higher than those in ASXL1(-) patients (P<0.05) . In the young group, the WBC of ASXL1(+) patients was higher than that of ASXL1(-) patients (z=-2.314, P=0.021) . ③IDH2 mutation and ASXL1 mutation was related (P=0.018, r=0.34) . In ASXL1(+) patients, the proportion of peripheral blasts in the high VAF group (VAF>40% ) was higher than that in the low VAF group (VAF<20% ) , and the proportion of aberrant nuclear cells was higher in the duplication and replacement mutation patients than in the deletion mutation patients (P<0.05) . ④The overall survival (OS) and progression-free survival (PFS) of ASXL1(+) patients were shorter than those of ASXL1(-) patients (median, 10 months vs 20 months, 10 months vs 17 months; P<0.05) . The proportion number of aberrant cells in nuclear cells (≥20% ) , complex karyotypes, and TET2 mutation were all independent risk variables that had an impact on the prognosis of ASXL1(+) patients, according to multivariate analysis (P<0.05) . Conclusion: ASXL1-mutated non-M(3) AML patients have higher WBC in peripheral blood, a higher proportion of aberrant cells in nuclear cells, lower CR(1) rate, and shorter OS and PFS. Additionally, a poor prognosis is linked to higher VAF, duplication, and substitution mutations in the ASXL1 gene, as well as the high proportion of aberrant cells in nuclear cells, complex karyotype, and TET2 mutation.

[Airborne pollens in Wuhan urban area and its correlation with hospital visits of allergic rhinitis patients from 2017 to 2018].

Objective: To investigate the species and concentrations of airborne pollens in Wuhan urban area and their correlation with the number of visits of allergic rhinitis patients. Methods: Retrospective analysis of pollen dispersal characteristics and the number of patients with allergic rhinitis presenting to Tongji Hospital of Huazhong University of Science and Technology in Wuhan city from October 2017 to September 2018, as well as pollen allergen testing results of patients with allergic rhinitis presenting to the Department of Allergy during the same period. Pollen data was collected by a 1-year air sampling conducted in Wuhan City during the same period using the volumetric method. The samples were examined microscopically to identify airborne pollen species and counted, and the concentrations of various pollens were calculated. Information on patients with allergic rhinitis who came to the hospital during the same period was collected, and the correlation between pollen concentration and the number of patient visits was statistically analyzed using Pearson correlation analysis. Results: A total of 35 types of airborne pollen were collected from October 2017 to September 2018. The dominant pollens in spring were Moraceae (68.46%, 1 042/1 522), Pendula (12.22%, 186/1 522) and Cupressaceae (2.30%, 35/1 522); in summer and autumn, the dominant pollens were Artemisia (3.81%, 58/1 522), Humulus (4.01%, 61/1 522) and Ambrosia (0.59%, 9/1 522). The peak number of visits for allergic rhinitis patients occurred in March-April and July-September, both exceeding 2 200 visits and reaching a maximum of 2 545 visits. There was a very weak correlation between the number of visits and the total pollen concentration (r=0.17, P=0.001). The average monthly pollen skin prick test positive rate of patients with allergic rhinitis was highest in March-May, exceeding 40% with a maximum of 45.73%, and there was a significant correlation between the positive pollen skin prick test positive rate and the average monthly pollen concentration (r=0.62, P=0.031). Conclusions: Pollen species and concentrations fluctuated continuously with time in Wuhan urban area, with peak pollen dispersal in spring from March to April and in autumn from August to September. The number of visits to patients with allergic rhinitis and the positive pollen skin prick test positive rate increased accordingly during the peak pollen concentration periods.

[The influence of peripheral blood sample storage and delivery on the quantitative detection result of BCR-ABL (P210) transcript levels].

Objective: To explore the influence of storage and delivery conditions of the peripheral blood samples from patients with chronic myeloid leukemia (CML) on the real-time quantitative PCR (RQ-PCR) detection of the BCR-ABL (P210) transcript levels. Methods: The peripheral blood samples of 84 CML patients were collected. The same sample was divided into different groups according to storage time (0, 6, 12, 24, 48, and 72 h) , temperature (room temperature, 18-24 ℃; low temperature, 2-8 ℃) , and vibration conditions (3, 6, and 12 h) . RQ-PCR was used to detect BCR-ABL (P210) transcript levels of the different groups. This study logarithmically transformed (log(10N)) the original data [BCR-ABL copy number, ABL copy number, and BCR-ABL (P210) transcript levels]. Results: ①Agarose gel electrophoresis showed significant RNA degradation of samples after storage for 48 and 72 h at room temperature. ②Among the overall samples, the BCR-ABL copy number of the samples stored at room temperature for 48 and 72 h was significantly lower than that of the samples stored at low temperature (P<0.05) . However, the BCR-ABL (P210) transcript levels had no significant difference between samples stored at low temperature and room temperature. ③No significant changes were noted in the BCR-ABL (P210) transcript levels at different storage times (6, 12, 24, 48, and 72 h) regardless of storage temperature (P>0.05) compared with that at baseline (0 h, -0.56±1.51) . ④ The BCR-ABL copy number of the overall sample only decreased significantly (P<0.05) at 48 h (2.93±1.59) and 72 h (2.79±1.42) compared with that at baseline (0 h, 3.35±1.60) when stored at room temperature. The ABL copy number in the overall sample decreased significantly at 48 and 72 h (whether low and room temperature; P<0.05) . However, no significant changes were noted in the BCR-ABL (P210) transcript levels after vibration for 3 h (-1.29±1.81) , 6 h (-1.24±1.72) , and 12 h (-1.18±1.68; P>0.05) compared with that at baseline (0 h, -0.60±1.37) . Conclusion: Sample storage time, storage temperature, and vibration can interfere with the results of BCR-ABL and ABL copy number but have no significant effect on the quantitative determination of BCR-ABL (P210) transcript levels. This study provides strong support for the feasibility of transregional transportation of peripheral blood samples from patients with CML.

[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

[Efficacy and safety of immunotherapy in dust mites andalternaria multi-sensitized allergic rhinitis patients].

Objective:The aim of this study is to evaluate the efficacy and safety of immunosuppression in patients with allergic rhinitis with multiple sensitization of dust mites and Alternaria.Method:An open, label random parallel controlled clinical study was conducted. Sixty dust mites and alternaria multi-sensitized allergic rhinitis patients were enrolled and randomized into immunotherapy group and medication group.Evaluation indicators included symptom scores, medication scores,symptom medication combined scores,RQLQ and serum allergen-specific IgE.In immunotherapy group, side effects were also observed and recorded.Result:After 24 months of treatment, all the scores were significantly lower than baseline,in both immunotherapy group and medication group.The scores of immunotherapy group were significantly lower than those of the medication group. Only local side effects were observed in immunotherapy group,without any systemic side effects and anaphylaxis.Conclusion: Mixed immunotherapy with dust mites and alternaria was effective and safe in allergic rhinitis patients and it had better curative effect than medication.

Antidiarrheal effect of bioactivity-guided fractions and bioactive components of pomegranate (Punica granatum L.) peels.

BACKGROUND: Pomegranate peels have been widely used to treat diarrhea in China. The antidiarrheal activities of aqueous extracts of pomegranate peels have been evaluated. However, there have not been any bioactivity-guided fractionation studies on the antidiarrheal effect to identify the bioactive components of the extract. METHODS: Bioactivity-guided fractionation of an aqueous extract of pomegranate peels was performed using different solvents of increasing polarity, generating fractions dissolved in ethyl acetate, n-butyl alcohol, and the residual fraction. The principal chemical composition of the active fraction was analyzed by HPLC/ESI-MS. KEY RESULTS: Fecal frequencies revealed that only the ethyl acetate fraction possessed significant antidiarrheal activity. Furthermore, administration of the ethyl acetate fraction at 100, 200, and 400 mg/kg significantly reduced gastrointestinal transit in charcoal meal tests in mice. It also significantly inhibited castor oil-induced enteropooling compared to control animals. Histopathological analysis revealed that small intestine lesions of mice treated with the ethyl acetate fraction were alleviated compared to those in mice treated with castor oil. The ethyl acetate fraction was found to be composed mainly of punicalagin, corilagin, and ellagic acid, and a combination of these compounds could mediate the antidiarrheal activities. CONCLUSION AND INFERENCES: Our study describes the protective effects of pomegranate peels against castor oil-induced diarrhea. The findings showed that the ethyl acetate fraction was the active fraction of pomegranate peels, of which punicalagin, corilagin, and ellagic acid were responsible for the antidiarrheal effect of aqueous extracts.

[The establishment and application of internal quality control system for real-time quantitative PCR detection of BCR-ABL (P210) transcript levels].

Objective: To set internal quality control system of BCR-ABL (P210) transcript levels for real-time quantitative PCR (RQ-PCR). Methods: Using K562 cells and HL-60 cells, we prepared high- and low-level BCR-ABL internal quality control substance. The BCR-ABL (P210) transcript levels of internal quality control substance have been determined for 184 times together with clinical samples from August 2013 to October 2015. The slope rate, intercept and correlation coefficient of standard curve were calculated according to different reagent lots (lots number 20130303, 20131212, 20140411 and 20150327 are called R1、R2、R3 and R4 for short respectively), and the detection results of quality control substance were calculated according to different reagent lots and quality control substance lots (lots number 20130725, 20140611 are called Q1、Q2 for short respectively). Then the results were analyzed by Levey-Jennings quality control chart combined with Westgard multi-rules theory. Results: ①We analyzed the slope rate and intercept of standard curve. Fifty-three times of the R1 reagent detection, 80 times of the R3 reagent detection and 14 times of the R4 reagent detection were all under control. For 37 times detection of R2 reagent, the slope rate was out of control for 6 times. It was lower than x-s for the 2-8 tests and upper the average for the 12-37 tests. The intercept was out of control for 9 times, upper the x+s for the 1-8 tests and lower the average for the 12-37 tests. ② According to the detection results of quality control substance, for Q1 quality control substance, 49 tests by R1 reagent were under control, and 1 out of 23 tests by R2 reagent was out of control. For Q2 quality control substance, 14 tests by R2 reagent detection, 72 tests by R3 reagent detection and 14 tests by R4 reagent were all under control. Conclusion: The preparation of high- and low-level quality control substance using K562 and HL-60 cells was convenient and the detection results were reliable and stable. The application of quality control substance combined with slope rate and intercept in the internal quality control may contribute to quality assurance for quantitative detection of BCR-ABL (P210) transcript levels.

[Study on high-efficiency gene transfer of pseudotyped HIV vector].

OBJECTIVE: To investigate the high-efficiency of pseudotyped HIV as gene transfer vector. METHODS: Three plasmids of pseudotyped HIV gene transfer vector system were transferred into packaging cell line 293T by Ca3 (PO4)2 precipitation method. GFP (Green Fluorescence Protein) or HSV-tk gene was constructed in the plasmid pHR'CS respectively (pHR'CS.GFP or pHR'CS.HSV-tk). The pseudotyped HIV particles were observed through electronic microscopy and were measured through spectrofluorophotometer. High titer pseudotyped HIV was harvested from volume of virus-producing cell supernatant and concentrated. Ovarian epithelial cancer cell line SKOV3 and normal human gingival fibroblast cell GF were infected by pseudotyped HIV. PCR and RT-PCR were resorted to demonstrate the successful transduction and transcription of the HSV-tk gene. After administration of GCV, the changes of those cells and apoptosis were observed through optical microscopy. The cytotoxicity efficacy of HSV-tk/GCV system was evaluated by MTT method. The growth inhibition rate (GIR) of cells and inhibition concentration 50 (IC50) were counted. RESULTS: The above plasmids were effectively transferred into 293T cell. A lot of pseudotyped HIV particles were observed through electronic microscopy. The virus supernatant had a high absorbing value at 510 nm through spectrofluorophotometer, which proved the existence of virus. After pseudotyped HIV infection, SKOV3 and GF had remarkable infection rate. 600 bp strand was seen through PCR and RT-PCR. Changes and apoptosis of cells followed by administration of GCV were observed. The MTT method showed that the cytotoxicity efficacy of GCV was high to SKOV3 and GF cell. CONCLUSIONS: The pseudotyped HIV is a high-efficiency gene transfer vector.