Objective:To explore the clinical application of preimplantation genetic testing for monogenic disorders (PGT-M) in an unique case with Spinal muscular atrophy (SMA) type 2+ 0.Methods:A special SMA family presented at the Third Affiliated Hospital of Guangzhou Medical University on October 19, 2020 was selected as the study subject. Multiple ligation-dependent probe amplification (MLPA) and molecular tagging linkage analysis were carried out to identify the SMN1 genotype of the couple and their fetus. Subsequently, next-generation sequencing (NGS), molecular tagging linkage analysis, and chromosomal microarray analysis were employed to determine the haplotypes and validate the result of PGT-M on the 11 embryos derived for the couple. Results:The female partner was identified as a carrier of the rare SMN1[2+ 0] variant, and prenatal diagnosis confirmed the fetus to be affected by SMA. Ultimately, PGT-M has successfully selected four embryos free from the pathogenic SMN1 variants and X chromosome deletion. Conclusion:PGT-M can effectively prevent the transmission of rare genetic variants such as the SMA 2+ 0 subtype in the families. Above finding has provided guidance for genetic counseling and family planning for the couple.

Objective To establish a PCR-based capillary electrophoresis(PCR/CE)to detect Survival Motor Neuron 1(SMN1)and Survival Motor Neuron 2(SMN2)genes and to evaluate its performance.Methods PCR/CE and Multiplex Ligation-dependent Probe Amplification(MLPA)for SMA gene diagnosis were used to blindly test the samples in sync.The performance of PCR/CE was assessed using MLPA results as the standard.Results A total of 336 samples were included in this study,consisting of 50 homozygous deletion types(14.9%),65 heterozygous deletion types(19.3%),and 221 non-deletion types(65.8%).The results of PCR/CE for detect-ing SMN1 and SMN2 copy numbers(0,1,2,3,≥4)were in complete agreement with the results of the MLPA.Conclusions PCR/CE for gene testing related to SMA could accurately detect copy numbers of exon 7 and exon 8 of the SMN1 and SMN2 genes(0,1,2,3,≥4).

Objective:To investigate the value of single sperm sequencing combined with PCR-reverse dot blot (PCR-RDB) technology in preimplantation genetic testing (PGT) of Southeast Asian deletion type α thalassemia.Methods:A couple of Southeast Asian deletion type α thalassemia carrier was selected in this case, who asked for assisted reproduction in the Department of Obstetrics and Gynecology of the Third Affiliated Hospital of Guangzhou Medical University on April 24, 2020. The male carrier was subjected to single sperm isolation by the swim-up method combined with micropipette due to his incomplete pedigree. Five single sperm samples were isolated and their whole-genome were amplified. The genotype of thalassemia of single sperm samples were determined by PCR-RDB. SNPs were selected as genetic markers in the range of 1 Mb upstream and downstream of -- SEA region to construct chromosomal haplotype. Next, we performed whole-genome amplification on six blastocyst biopsy samples, and then next-generation sequencing was carried out to detect the carrier status of the embryos. Finally, the non-pathogenic blastocysts were selected for transplantation, and the preimplantation genetic testing for monogenic (PGT-M) results were confirmed by the prenatal genetic diagnosis during pregnancy. Results:The pathogenic variant in the female was inherited from her mother, and four of the five single sperm samples were wild-type. Ten male informative SNP markers were identified by single sperm sequencing, and six female informative SNP markers were identified by linkage analysis of female family. PGT-M results indicated that four blastocysts were αα/-- SEA and two were -- SEA/-- SEA. The result of prenatal diagnosis revealed that the fetus was a carrier of -- SEA , which was consistent with PGT-M result, and a healthy girl was delivered at 40 weeks of gestation. Conclusion:For male carriers of Southeast Asian deletion type α thalassemia with incomplete pedigree, single sperm sequencing combined with PCR-RDB technology can be used to select SNP sites, and PGT-M can be performed by linkage analysis.

Objective:To investigate the value of single sperm sequencing combined with PCR-reverse dot blot (PCR-RDB) technology in preimplantation genetic testing (PGT) of Southeast Asian deletion type α thalassemia.Methods:A couple of Southeast Asian deletion type α thalassemia carrier was selected in this case, who asked for assisted reproduction in the Department of Obstetrics and Gynecology of the Third Affiliated Hospital of Guangzhou Medical University on April 24, 2020. The male carrier was subjected to single sperm isolation by the swim-up method combined with micropipette due to his incomplete pedigree. Five single sperm samples were isolated and their whole-genome were amplified. The genotype of thalassemia of single sperm samples were determined by PCR-RDB. SNPs were selected as genetic markers in the range of 1 Mb upstream and downstream of -- SEA region to construct chromosomal haplotype. Next, we performed whole-genome amplification on six blastocyst biopsy samples, and then next-generation sequencing was carried out to detect the carrier status of the embryos. Finally, the non-pathogenic blastocysts were selected for transplantation, and the preimplantation genetic testing for monogenic (PGT-M) results were confirmed by the prenatal genetic diagnosis during pregnancy. Results:The pathogenic variant in the female was inherited from her mother, and four of the five single sperm samples were wild-type. Ten male informative SNP markers were identified by single sperm sequencing, and six female informative SNP markers were identified by linkage analysis of female family. PGT-M results indicated that four blastocysts were αα/-- SEA and two were -- SEA/-- SEA. The result of prenatal diagnosis revealed that the fetus was a carrier of -- SEA , which was consistent with PGT-M result, and a healthy girl was delivered at 40 weeks of gestation. Conclusion:For male carriers of Southeast Asian deletion type α thalassemia with incomplete pedigree, single sperm sequencing combined with PCR-RDB technology can be used to select SNP sites, and PGT-M can be performed by linkage analysis.

Objective To explore the forensic pathological characteristics and the main identification points of fatal cardiac tamponade. Methods 38 cases of fatal cardiac tamponade from department of pathology, the first affiliated hospital of chengdu university of TCM from 2005 to 2015 were reviewed and analyzed retrospectively. Results Fatal cardiac tamponade mostly occurred to men (71.1%) with an average age of 44; Bloody effusion accounted for 85% of the direct causes of death (34 cases). The most underlying causes of death were diseases (73.7%), majorly aortic dissection, coronary heart disease and malignant tumors. Seventy five percent of death occurred within 12 hours of illness. Medical behaviors were involved in 30 cases (78.9%), of which 26 cases (86.7%)were without medical malpractice. The relationship between injury and disease was involved in 15 fatal cases (39.5%). Conclusion The basic requirement for accurately completing forensic medical appraisal of fatal cardiac tamponade cases was to master the forensic pathological characteristics and the path of forensic identification.

BACKGROUND: 5-azacytidine (5-Aza) has been frequently used to induce bone marrow mesenchymal stem cells (BMSCs)differentiation into cardiomyocyte.OBJECTIVE: To observe expression of cardiomyocyte-related receptors in cardiomyogenic differentiation of rat BMSCs.METHODS: BMSCs of passage three were assigned to four groups: group Ⅰ: L-DMEM solution alone was replaced; Ⅱ:L-DMEM solution was replaced after induction of 100 mg/L AST+5 μmol/L 5-Aza for 24 hours; group Ⅲ: L-DMEM solution wasreplaced after induction of 10 μmol/L 5-Aza for 24 hours; and group Ⅳ: L-DMEM solution was replaced after induction of 5 μmol/L5-Aza for 24 hours. Culture medium was replaced every 3 days in each group. Differentiated cells were identified after 30 days ofinduction.RESULTS AND CONCLUSION: Expression of cardiomyocyte specific proteins Nkx2.5, cTnT and Desmin was detected in groupsⅢ, Ⅳ and Ⅱ after induction compared with group Ⅰ , with significant differences (P < 0.01). The amount of cTnT and Desminexpression expression was significantly higher in groups Ⅱ and Ⅲ compared with group Ⅳ (P < 0.01). The level of Nkx2.5expression was significantly higher in groups Ⅱ (P < 0.01) and Ⅲ (P < 0.05) compared with group Ⅳ. No Nkx2.5, cTnT andDesmin espression was detected in group Ⅰ. After induction for 2 weeks, cells with spontaneous contractility were observed ingroups Ⅱ and Ⅲ, indicating differentiation towards cardiomyocyte after induction. Results demonstrated that induction effectswere similar between 100 mg/L AST+5 μmol/L 5-Aza and 10 μmol/L 5-Aza. This may contribute to cytoprotective effects of AST,which can promote vascular endothelial cell proliferation, enhance celss tolerance to 5-Aza-induced cytotoxicity and upregulatecardiac-specific protein expression.