Next-generation sequencing has revolutionized family-based association tests for rare variants. As the lower power of genome wide association study for detecting casual rare variants, methods aggregating effects of multiple variants have been proposed, such as burden tests and variance component tests. This paper summarizes the methods of rare variants association test that can be applied for family data, introduces their principles, characteristics and applicable conditions and discusses the shortcomings and the improvement of the present methods.
Computer Simulation ; Family Relations ; Genetic Association Studies ; Genetic Variation ; Genome-Wide Association Study/methods* ; Humans

OBJECTIVE: To explore the association between de novo mutations (DNM) and non-syndromic cleft lip with or without palate (NSCL/P) using case-parent trio design. METHODS: Whole-exome sequencing was conducted for twenty-two NSCL/P trios and Genome Analysis ToolKit (GATK) was used to identify DNM by comparing the alleles of the cases and their parents. Information of predictable functions was annotated to the locus with SnpEff. Enrichment analysis for DNM was conducted to test the difference between the actual number and the expected number of DNM, and to explore whether there were genes with more DNM than expected. NSCL/P-related genes indicated by previous studies with solid evidence were selected by literature reviewing. Protein-protein interactions analysis was conducted among the genes with protein-altering DNM and NSCL/P-related genes. R package "denovolyzeR" was used for the enrichment analysis (Bonferroni correction: P=0.05/n, n is the number of genes in the whole genome range). Protein-protein interactions among genes with DNM and genes with solid evidence on the risk factors of NSCL/P were predicted depending on the information provided by STRING database. RESULTS: A total of 339 908 SNPs were qualified for the subsequent analysis after quality control. The number of high confident DNM identified by GATK was 345. Among those DNM, forty-four DNM were missense mutations, one DNM was nonsense mutation, two DNM were splicing site mutations, twenty DNM were synonymous mutations and others were located in intron or intergenic regions. The results of enrichment analysis showed that the number of protein-altering DNM on the exome regions was larger than expected (P < 0.05), and five genes (KRTCAP2, HMCN2, ANKRD36C, ADGRL2 and DIPK2A) had more DNM than expected (P < 0.05/(2×19 618)). Protein-protein interaction analysis was conducted among forty-six genes with protein-altering DNM and thirteen genes associated with NSCL/P selected by literature reviewing. Six pairs of interactions occurred between the genes with DNM and known NSCL/P-related genes. The score measuring the confidence level of the predicted interaction between RGPD4 and SUMO1 was 0.868, which was higher than the scores for other pairs of genes. CONCLUSION Our study provided novel insights into the development of NSCL/P and demonstrated that functional analyses of genes carrying DNM were warranted to understand the genetic architecture of complex diseases.
Asians ; Case-Control Studies ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Genotype ; Humans ; Mutation ; Parents ; Polymorphism, Single Nucleotide ; Whole Exome Sequencing

BACKGROUND: A patient's infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence. METHODS: The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients' oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed. RESULTS: In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0-62.0) years were analyzed. After in-hospital treatment, patients' inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0-11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients' stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0-16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0-4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients' urine specimens after throat swabs were negative. Using a multiple linear regression model (F = 2.669, P = 0.044, and adjusted R = 0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients' stools (t = -2.699, P = 0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs. 8.0 days, respectively; t = 2.550, P = 0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs. 11 days, respectively; t = 4.631, P < 0.001). There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results (P > 0.05). CONCLUSIONS In brief, as the clearance of viral RNA in patients' stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.
Adult ; Aged ; Betacoronavirus ; genetics ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; genetics ; rehabilitation ; Female ; Humans ; Male ; Middle Aged ; Pandemics ; Pneumonia, Viral ; genetics ; rehabilitation ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; Retrospective Studies

Background: A patient’s infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence. Methods: The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients’ oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed. Results: In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0–62.0) years were analyzed. After in-hospital treatment, patients’ inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0–11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients’ stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0–16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0–4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients’ urine specimens after throat swabs were negative. Using a multiple linear regression model (F=2.669, P=0.044, and adjusted R²=0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients’ stools (t=-2.699, P=0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs 8.0 days, respectively; t=2.550, P=0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs 11 days, respectively; t=4.631, P <0.001). There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results (P >0.05). Conclusions In brief, as the clearance of viral RNA in patients’ stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.

Objective: To discuss the effect of Gandou decoction (GDD) on the immune index of spleen in TX mice of Wilson's disease model. Method: The mice were divided into normal group, model group and GDD or tetrathiomolybdate(TM)treatment group, with 20 mice in each group. Each group was fed in various ways for 30 successive days. Normal group:10 normal DL mice were randomly selected and feed normally. Model group:20 TX mice were randomly selected and feed with 2 mL·kg^-1·d^-1ig saline by gavage twice per day. GDD or TM treatment group:80 TX mice were randomly selected and feed with 2 mL·kg^-1·d^-1 ig Gandou decoction 22,44,66 g·kg^-1 or tetrathiomolybdate by gavage twice per day. ICP-MS was used to compare the expressions of trace elements inside the mice's spleens, flow cytometry was applied to detect the mice T lymphocyte subsets of splenic tissue CD4⁺, CD8⁺, CD4⁺/CD8⁺, and Western blot was used to detect the expressions of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-17 (IL-17) and interleukin-18 (IL-18). Result: Flow ICP-MS results showed that GDD can reduce Cu of mice's spleen, flow cytometry results showed that CD4⁺and CD8⁺in model group were increased than those in normal group (P<0.01), and CD4⁺/CD8⁺was decreased (P<0.01); compared with model group, CD4⁺and CD8⁺in middle and high-dose GDD groups were decreased (P<0.01), and CD4⁺/CD8⁺was increased. According to Western blot detection, compared with normal group, the expressions of IL-2, IL-8, IL-17, IL-18, TNF-α and IFN-γ in the model group were increased (P<0.01); compared with model group, the expressions of TNF-α, IFN-γ, IL-2, IL-8, IL-17 and IL-18 in the GDD middle and high or TM group were decreased (P<0.05, P<0.01). Compared with model group, the expressions of IL-2, IL-8, IL-17 and TNF-α in the GDD low were decreased (P<0.05). Conclusion: Spleen of TX mice shows the cellular immunity hyperfunction, which is mainly dominated by the negative immunoloregulation. GDD has a certain effect in regulating cellular immunity hyperfunctional state of TX mice, but it's difficult to thoroughly change the negative immune regulation.

ObjectiveTo explore the effect of Modified Dahuang Zhechong Granule (MDZG) on the development and maturation of epididymal sperm in experimental varicocele (VC) rats.

METHODSSixty SD male rats were randomly divided into six groups of equal number, sham operation, VC model, Aescuven forte, and low-, medium- and high-dose MDZG. The model of left VC was made by the Turner method in all the rats except those of the sham operation group, followed by treatment with 0.9% normal saline for the animals in the sham operation and VC model groups, Aescuven forte tablets at 54 mg per kg of the body weight for those in the Aescuven forte group, and MDZG at 0.6, 1.2 and 2.4 g/ml for those in the low-, medium- and high-dose MDZG groups, all administered intragastrically qd for 8 successive weeks. Then, all the rats were sacrificed and their left epididymides harvested for examination of the quality of the epididymal sperm and the local microscopic and ultrastructural changes of the epididymal tissue.

RESULTSThe VC model rats showed significant apoptosis of the epididymal sperm cells, interstitial edema, microvascular dilatation, degeneration and degeneration of the epithelial cells, degeneration of some principal cells and basal cell vacuoles, and immature spermatids in the lumen. Sperm motility was significantly increased in the Aescuven forte and low-, medium- and high-dose MDZG groups as compared with the VC models (P <0.01). Both sperm concentration and motility were markedly higher in the high-dose MDZG than in the Aescuven forte group (P <0.05). Remarkable apoptosis of epididymal sperm cells was observed in the microenvironment of sperm development in the VC models, which exhibited no statistically significant difference from that in the rats of the medium- and high-dose MDZG groups.

CONCLUSIONSExperimental varicocele induced local apoptosis of epididymal sperm cells, interstitial edema and microvascular dilatation in the rat epididymis, while Modified Dahuang Zhechong Granule could improve the stability of epididymal sperm maturation and contribute to their development.

Aesculus ; chemistry ; Animals ; Apoptosis ; Drugs, Chinese Herbal ; pharmacology ; Edema ; chemically induced ; Epididymis ; drug effects ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Sperm Motility ; drug effects ; Spermatozoa ; cytology ; drug effects ; Varicocele ; chemically induced ; drug therapy ; pathology

Objective To design a self-service terminal for hyperbaric oxygen therapy in order to improve the orderliness and efficiency of hyperbaric oxygen therapy.Methods The hardware included mainframe and cabinet,and the software used human-computer interface.Network database was connected with ADO technology,and Delphi 7.0,Pascal compiling code were applied to code compilation.Results The self-service terminal involved the patient into the information chain of hyperbaric oxygen therapy to enhance the working efficiency and therapy orderliness.Conclusion The terminal behaves well in hardware,software,network database and human-computer interaction,and thus is worthy promoting to medium and large hyperbaric oxygen chambers.

Objective To design and develop an information management system based on network database for hyperbaric oxygen therapy to solve the problems in repeated operation,excessive data,copying and etc.Methods Network database was used to establish the information storage unit,design software and hardware architecture as well as develop information treatment units for medical terminal,self service,hand-hold intelligent terminal,appointment App program and etc.Results The system realized rapid printing and recognition of patient information,quick response of server,high accuracy of charging module,and high efficiency of self service terminal and appointment registration.Conchusion The system optimizes hyperbaric oxygen therapy flow,enhances working efficiency of medical staffs and gains high patient satisfaction,and thus is worthy promoting clinically.

OBJECTIVETo assess the clinical features and therapeutic effect of free thin anterolateral thigh flap for the reconstruction of intraoral defects.

METHODSThe clinicopathologic data of 34 cases with oral carcinoma were obtained from Institute and Hospital of Stomatology, Nanjing University Medical School, from December 2008 to December 2011. These 34 patients underwent the simultaneous tumor resection and intraoral defects reconstruction with free thin anterolateral thigh flaps. The defects were located at tongue, buccal, mouth floor, and so on. The subcutaneous fat thickness of the flap at the site of the perforator is usually measured by ultrasound before the operation. If the thickness of the subcutaneous fat at the site of the perforator exceeded 1.5 cm, the patient was excluded from the study.

RESULTSThere were 16 male and 18 female patients. The mean age was 55.4 years. Among the 34 patients, 26 flaps with musculocutaneous perforators and 8 flaps with septo-cutaneous perforators were used. Partial flap necrosis occurred in 2 patients.

CONCLUSIONSThe free thin anterolateral thigh flap is the ideal soft tissue flap for the intraoral defects reconstruction. Satisfactory functional results can he achieved at recipient area with minimal morbidity at the donor site area.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Mouth Neoplasms ; surgery ; Reconstructive Surgical Procedures ; methods ; Skin Transplantation ; methods ; Soft Tissue Injuries ; etiology ; surgery ; Surgical Flaps ; Thigh

OBJECTIVETo assess the clinical features and therapeutic efficacy of anterolateral thigh (ALT) flaps for the intraoral defects reconstruction.

METHODSThe clinicopathologic data of 67 cases with oral tumors were obtained from School of Stomatology, Nanjing University Medical Center from Dec. 2008 to Dec. 2010. All the patients underwent the simultaneous tumor resection and intraoral defects reconstruction with free anterolateral thigh flaps. The defects included the tongue, buccal, gingival, mouth floor, and so on. The descending branch of lateral femoral circumflex artery was anastomosed to the external maxillary artery or superior thyroid artery; the vein was anastomosed to the common facial vein or external jugular vein. The flaps were divided into three types: musculocutaneous ALT flap, fasciocutaneous ALT flap and thinned ALT flap.

RESULTSThere were 38 male patients and 29 female. The anterolateral thigh flaps included 35 musculocutaneous flaps, 17 fasciocutaneous flaps and 15 thinned flaps. The success rate was 98.5% (66/67). Partial necrosis happened in one case with diabetes, which healed after debridement and dressing. 1 flap was totally necrosis. Double venous anastomosis was performed in 41 flaps, and one venous anastomosis was performed in 26 flaps. 8 patients required operative exploration in the perioperative period including 6 flaps with thrombotic events (5 flaps were complete survival after the salvages, and 1 flap was failure) , 1 flap with hematoma, and 1 flap with twisting of perforator. The follow-up period ranged from 2 to 24 months( mean, 8.7 months). The result was satisfied. The donor sites were closed directly in all patients, and the wounds healed uneventfully.

CONCLUSIONSThe free anterolateral thigh flap is an ideal soft tissue flap for the intraoral defects reconstruction with good functional result at recipient area and less morbidity at the donor site.

Adult ; Aged ; Female ; Free Tissue Flaps ; Humans ; Male ; Middle Aged ; Mouth Neoplasms ; surgery ; Reconstructive Surgical Procedures ; methods ; Skin Transplantation ; methods ; Thigh ; surgery