Identification and expression analysis of the YABBY gene family in strawberry / 生物工程学报

YABBY proteins are important transcription factors that regulate morphogenesis and organ development in plants. In order to study the YABBY of strawberry, bioinformatic technique were used to identify the YABBY gene families in Fragaria vesca (diploid) and Fragaria×ananassa (octoploid), and then analyze the sequence characters, phylogeny and collinearity of the family members. The RNA-seq data and the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique were used to assay the expression patterns of the family members. A green fluorescent protein (GFP) was fused with FvYABBYs and transiently expressed in tobacco leaf cells for the subcellular localization. As the results, six FvYABBY genes and 26 FxaYABBY genes were identified from F. vesca and F.×ananassa, respectively. The FvYABBY genes were grouped into five clades, and five family members were orthologous with AtYABBY genes of Arabidopsis. In F. vesca, all of the FvYABBYs were basically not expressed not expressed in root and receptacle, while FvYABBY1, FvYABBY2, FvYABBY5 and FvYABBY6 were highly expressed in leaf, shoot, flower and achene. In F.×ananassa, FxaYABBY1, FxaYABBY2, FxaYABBY5 and FxaYABBY6 were expressed in achene, and all FxaYABBY were poorly or not expressed in receptacle. Additionally, under the abiotic stresses of low temperature, high salt and drought, the expression of FvYABBY1, FvYABBY3, FvYABBY4 and FvYABBY6 were down-regulated, FvYABBY5 was up-regulated, and FvYABBY2 was up-regulated and then down-regulated. In tobacco leaf cells, the subcellular localization of FvYABBY proteins were in the nucleus. These results provides a foundation for the functional researches of YABBY gene in strawberry.

Molecular analysis of the new allele 803delC of subtype B / 中国输血杂志

【Objective】 To analyze the serological characteristics and molecular mechanism of a novel B subtype allele 803delC. 【Methods】 ABO blood group was detected by serological method. Sequence-specific primer polymerase chain reaction (PCR-SSP) was used to detect ABO blood group genes. The coding region of exon 1-7 of ABO gene was detected by Sanger sequencing to determine the mutation site. 【Results】 Serological identification of patients was with forward O-type and reverse B-type. The result of PCR-SSP genotyping was A/O. There was A gene, which was not consistent with serological results. Further Sanger double-strand sequencing revealed that the C-base was deleted at position 803 of exon 7 on the basis of ABO*B. 01/ABO*O. 01.01. The mutation eventually leads to the amino acid substitution of p. Ala268Gly and p. Phe269Ser and the production of new open reading frame starting at position 269, with the new open reading frame No.20 amino acid being stop codon, resulted in the termination of B gene expression. Further single-strand sequencing of the ABO gene revealed that the mutation was located in the ABO*B. 01 gene. The mutation was submitted to the NCBI database with the number OR343908. 【Conclusion】 A new ABO allele leading to B variant has been found in Chinese population. Genetic detection can be used to identify the ambiguous blood group with discrepancy between forward and reverse blood grouping.

Molecular epidemiology and antifungal susceptibilities of Aspergillus species isolated from patients with invasive aspergillosis

SUMMARY OBJECTIVE: The aim of this study was to evaluate the demographic data, molecular epidemiology, and in vitro antifungal susceptibility results of patients with Aspergillus isolated from various clinical specimens. METHODS: A total of 44 Aspergillus strains were studied. The definition of invasive aspergillosis in patients was made according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Strains were phenotypically and molecularly identified. Demographic characteristics of patients and genotypes of strains were evaluated. Phylogenetic analysis was done by the The Unweighted Pair-Group Method with Arithmetic Mean (UPGMA). Antifungal susceptibility of strains was determined according to The Clinical and Laboratory Standards Institute (CLSI)-M61-Ed2 and The European Committee on Antimicrobial Susceptibility Testing (EUCAST). RESULTS: A total of 11 patients were classified as proven and 33 as probable invasive aspergillosis. There was a statistically significant difference in age groups, subdisease, neutropenic, and receiving chemotherapy between groups. A total of 23 strains were identified as Aspergillus fumigatus, 12 as Aspergillus niger, 6 as Aspergillus flavus, and 3 as Aspergillus terreus. Phylogenetic analysis revealed five different genotypes. No statistical difference was found in the comparisons between patients groups and genotype groups. There was a statistically significant difference between genotype groups and voriconazole, posaconazole, and itraconazole Minimum Inhibition Concentration (MIC). CONCLUSION: Accurate identification of strains and antifungal susceptibility studies should be performed due to azole and amphotericin B resistance. Genotyping studies are important in infection control due to identifying sources of infection and transmission routes.

Genotyping identification and sequence analysis of ABO ambiguous blood group in 20 cases / 中国输血杂志

【Objective】 To study the genotypes of ABO ambiguous blood group samples(n=20) and identify their molecular biological characteristics. 【Methods】 The serological phenotype of the samples was analyzed by serological techniques. Seven exons of ABO gene were amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced; the genotypes and sequences of ABO subtypes were analyzed. 【Results】 The serological phenotypes of 20 samples presenting ABO ambiguous blood group were as follows: weak A antigen (n=5), weak A antigen combined with anti-A1 antibody (n=5), normal A antigen combined with anti-A1 antibody (n=2), weak B antigen (n=8). The genotypes of them were as follows: Ax02/O01 (n=3), Ael07/O01 (n=2), B313/O01 (n=2), A204/O02 (n=1), A220/O01 (n=1), Ael07/O02 (n=1), Ael02/O01 (n=1), Ael02/O02 (n=1), Ax03/O01 (n=1), Ax03/O02 (n=1), B313/O02 (n=1), B302/O01 (n=1), B302/O02 (n=1), Bw19/O02 (n=1), A102/B313 (n=1) and A101/Bw37 (n=1). 【Conclusion】 ABO genotyping technology can accurately identify the ambiguous blood group of samples, provide definite genetic information of blood group and ensure the safety of clinical transfusion.

Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection / 生物工程学报

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

Mitochondrial genome sequence characteristics and phylogenetic analysis of Schizothorax argentatus / 生物工程学报

Schizothorax argentatus that only distributes in the Ili River basin in Xinjiang is one of the rare and endangered species of schizothorax in China, thus has high scientific and economic values. In this study, the complete mitochondrial genome sequence of S. argenteus with a length of 16 580 bp was obtained by high-throughput sequencing. The gene compositions and arrangement were similar to those of typical vertebrates. It contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding region (D-loop). The nucleotide compositions were A (30.25%), G (17.28%), C (27.20%), and T (25.27%), respectively, showing obvious AT bias and anti-G bias. Among the tRNA genes, only tRNA-Ser(GCU) could not form a typical cloverleaf structure due to the lack of dihydrouracil arm. The AT-skew and GC-skew values of the ND6 gene were fluctuating the most, suggesting that the gene may experience different selection and mutation pressures from other genes. The mitochondrial control region of S. argenteus contained three different domains, i.e., termination sequence region (ETAS), central conserved region (CSB-F, CSB-E, CSB-D, and CSB-B), and conserved sequence region (CSB1, CSB2, and CSB3). The conserved sequence fragment TT (AT) nGTG, which was ubiquitous in Cypriniformes, was identified at about 50 bp downstream CSB3. Phylogenetic relationships based on the complete mitochondrial genome sequence of 28 Schizothorax species showed that S. argenteus had differentiated earlier and had a distant relationship with other species, which may be closely related to the geographical location and the hydrological environment where it lives.

Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province / 中国中药杂志

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

Application of single-cell RNA sequencing in melanoma research / 中华皮肤科杂志

Tumor heterogeneity is one of the important characteristics of melanoma. Single-cell RNA sequencing not only can further reveal the heterogeneity of melanoma cells, but also has unique advantages in analyzing the occurrence and development of melanoma, finding new targets for immunotherapy and uncovering mechanisms of drug resistance. This review summarizes the application of single-cell RNA sequencing in melanoma research.

Postoperative infection after anterior cruciate ligament reconstruction caused by Finegoldia magna: a case report / 中华骨科杂志

One case of knee infection after anterior cruciate ligament reconstruction caused by the gram-positive anaerobic bacterium Finegoldia magna was reported. The patient was admitted to hospital due to fever and knee joint swelling and pain after anterior cruciate ligament reconstruction. Through medical history, physical examination, imaging examination and next-generation sequencing, it was confirmed that the infection was caused by Finegoldia magna. Through literature review, 37 literatures on infectious diseases caused by Finegoldia magna was retrieved and analyzed, and the identification points of anaerobic bacteria, the application of second-generation sequencing technology and the treatment status of infection after anterior cruciate ligament reconstruction were reviewed. The incidence of infection after arthroscopic anterior cruciate ligament reconstruction is low, while anaerobic infection is even more rare and difficult to culture. The next-generation sequencing can be used to assist the diagnosis. On the basis of giving priority to the preservation of the reconstructed ligament, the combined use of arthroscopic debridement, irrigation and sensitive antibiotics is the main treatment method.

Circulation of human respiratory syncytial virus and new ON1 genotype in northern Viet Nam, 2017–2020

@#Objective: Human respiratory syncytial virus (RSV) is a primary cause of paediatric severe acute respiratory infection (SARI) worldwide, especially in developing countries. We investigated the genetic characteristics of RSV in northern Viet Nam to determine the prevalence and distribution of subtypes as well as the diversity and transmission patterns of genotypes. Methods: In two facilities, from January 2017 to December 2020, 1563 clinical specimens were collected from paediatric patients hospitalized with SARI and tested for RSV. Selected positive samples underwent sequencing analysis targeting the second hypervariable region of the G gene using next-generation sequencing. Results: The RSV positivity rate was 28.02% (438/1563 samples), and prevalence was highest in children aged <1 year (43.84%; 192/438). Subtype RSV-A accounted for 53.42% (234/438) of cases, RSV-B for 45.89% (201/438), and there was coinfection in 0.68% (3/438). Both subtypes cocirculated and peaked during August–September in each year of the study. Phylogenetic analysis showed that RSV-A samples belonged to the ON1 genotype, which has three subgenotypes: ON1.1, ON1.2 and ON1.3. However, we did not find the 72-nucleotide duplication in the second hypervariable region of the G gene, a characteristic of genotype ON1, in any RSV-A samples. RSV-B samples belonged to genotype BA9. Discussion: Our results provide additional molecular characterization of RSV infections in Viet Nam. Specially, our study is the first to report the absence of the 72-nucleotide duplication in the G gene of RSV-A genotype ON1 in Viet Nam, which may help in understanding the genetic evolution of RSV and be useful for vaccine development in the future.

Phylogenetic analysis of chloroplast genome of Tussilago farfara L / 药学学报

italic>Tussilago farfara L. is a perennial herb of Tussilago genus in the Compositae family. Its dried buds and leaves have good biological activities and have a long history of medicinal use in China and Europe. In this paper, we investigated the whole chloroplast genome characteristics, sequence duplication, structural variation and phylogeny of the Tussilago farfara L. After sequencing the Tussilago farfara L. chloroplast genome using Illumination technology, the complete Tussilago farfara L. chloroplast genome was further obtained by assembly and annotation, followed by a series of inverted repeat-large single copy/small single copy region contraction and expansion analysis, genome sequence variation, etc. The sequences of 13 homologous plants downloaded from NCBI were used to construct a neighbor-joining phylogenetic tree. The results showed that the total GC content of the chloroplast genome was 37.4% and the length was 150 300 bp; 125 genes were annotated, including 82 protein-coding genes, 35 tRNAs and 8 rRNAs; 148 (simple sequence repeats, SSR) loci were detected, and the relative synonymous codon usage showed that 31 codons out of 64 codons had a usage of >1. In the phylogenetic analysis, the chloroplast genomes of the seven species of Asteraceae, including the Yulin Tussilago farfara L., were highly conserved, and the sequence variation of the (large single-copy, LSC) and (small single-copy, SSC) regions was higher than that of the (inverted repeat, IR) region. This is in general agreement with the reported phylogeny of Yulin Tussilago farfara L. In this study, we obtained a high quality chloroplast genome and analyzed its genome characteristics, codon preference, SSR characteristics, SC/IR boundary, sequence variation and phylogeny, which can provide a basis for species identification, genetic diversity analysis and resource development of this medicinal plant.

Application of targeted capture high depth sequencing and transcriptome sequencing in childhood B-cell acute lymphoblastic leukemia / 白血病·淋巴瘤

Objective:To investigate the differences among targeted capture high depth sequencing (Panel-seq), transcriptome sequencing (RNA-seq) and traditional detection methods in cytogenetic and molecular genetic typing of childhood B-cell acute lymphoblastic leukemia (B-ALL) and their significances.Methods:The clinical data of 152 newly diagnosed childhood B-ALL cases in Guangzhou Women and Children's Medical Center from September 2020 to December 2021 were retrospectively analyzed. Along with traditional cytogenetic and molecular detection methods including karyotyping, fluorescence in situ hybridization (FISH) and 43 kinds of fusion gene quantitative screening for traditional cells and molecular genetic detection, both Panel-seq and RNA-seq were also performed. Panel-seq covered more than 600 genes with common mutations in hematological tumors, from which fusion genes and gene mutations were both analyzed. RNA-seq was used to analyze fusion genes, gene mutations, gene expression, and copy number variation at the chromosome level. High hyperdiploid karyotype was estimated by using gene expression profile clustering and copy number variations. The cytogenetic typing results of all detection methods were also analyzed.Results:Among 152 patients, 93 cases were males and 59 cases were females, with the median age of 4.0 years (0.8-13.0 years). The median blast cell ratio was 0.855 (0.215-0.965). The traditional detection methods could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusion, 27 cases (17.8%) with ETV6-RUNX1, 1 case (0.7%) with iAMP21, 5 cases (3.3%) with MLL rearrangement, 8 cases (5.3%) with TCF3-PBX1 and 22 cases (14.5%) with high hyperdiploid karyotype. Panel-seq could identify 4 cases (2.6%) with BCR-ABL1, 2 cases (1.3%) with CRLF2 gene-related fusions, 27 cases (17.8%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 5 cases (3.3%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 8 cases (5.3%) with TCF3-PBX1 fusions, 4 cases (2.6%) with ZNF384 gene-related fusions, and 2 cases (1.3%) with IKZF1 N159Y mutations. Among 152 patients, 1 case with MLL rearrangement didn't receive RNA-seq detection because of sample quality; in other 151 B-ALL cases, 1 case (0.7%) with ACIN1-NUTM1, 4 cases (2.6%) with BCR-ABL1, 3 cases (2.0%) with CRLF2 gene-related fusions, 8 cases (5.3%) with DUX4 gene-related fusions, 27 cases (17.9%) with ETV6-RUNX1, 3 cases (2.0%) with MEF2D gene-related fusions, 1 case (0.7%) with MEIS1-FOXO1, 4 cases (2.6%) with MLL rearrangement, 5 cases (3.3%) with PAX5 gene-related fusions, 1 case (0.7%) with ZMIZ1-ABL1, 8 cases (5.3%) with TCF3-PBX1,4 cases (2.6%) with ZNF384 gene-related fusions, 61 cases (40.4%) with hyperdiploid karyotypes, and 2 cases (1.3%) with IKZF1 N159Y mutations were detected; RNA-seq had obvious advantage in detecting fusion gene and hyperdiploid karyotype. The cytogenetic and molecular genetic typing rates of traditional method, Panel-seq and RNA-seq were 45.4% (69/152), 40.1% (61/152) and 87.4% (132/151), respectively. The combination of the three could identify 89.5% (136/152) of childhood B-ALL patients.Conclusions:The combination of Panel-seq and RNA-seq can increase the detection rate of genetic abnormality in childhood B-ALL, which provides a more accurate molecular genetic classification for B-ALL and the basis for treatment guideline and prognosis judgement.

Analysis of circRNA in esophageal squamous cell carcinoma based on high-throughput sequencing data / 国际肿瘤学杂志

Objective:To analyze circRNAs specifically differentially expressed in esophageal squamous cell carcinoma (ESCC) based on high-throughput sequencing data.Methods:Six patients with pathologically confirmed ESCC in Tangdu Hospital of Air Force Medical University from March 2018 to March 2019 were selected as the research subjects, among which 3 were stage Ⅰ ESCC and 3 were stage Ⅲ ESCC. High-throughput sequencing technology was used to analyze the difference in the expression of circRNA in cancer tissues and adjacent tissues of patients. GO enrichment analysis, KEGG enrichment analysis and Venn analysis were performed on differentially expressed genes. The circRNA-miRNA-mRNA network was constructed using Cytoscape software. The most significantly differentially expressed genes in cancer tissues were verified in cells and tissues, and the relationships between circRNAs and clinical pathological indicators of patients were analyzed.Results:A total of 553 differentially expressed circRNAs were screened in paracancerous tissues and cancer tissues of 3 stage Ⅰ ESCC patients, of which 413 were up-regulated and 140 were down-regulated in cancer tissues; A total of 425 differentially expressed circRNAs were screened in paracancerous tissues and cancer tissues of 3 stage Ⅲ ESCC patients, of which 276 were up-regulated and 149 were down-regulated in cancer tissues. GO enrichment analysis showed that the host genes of differential circRNAs in patients with stage Ⅰ ESCC were mainly enriched in cell cycle-related biological processes such as mitotic G 2/M transition. The host genes of differential circRNAs in patients with stage Ⅲ ESCC were mainly enriched in biological processes related to cell division and tumor development, such as mitotic spindle checkpoint and cell matrix adhesion. KEGG enrichment analysis showed that the differential circRNAs in cancer tissues of stage Ⅰ and stage Ⅲ ESCC patients were mainly enriched in cancer-related biological pathways such as cell adhesion. The results of Venn analysis showed that in stage Ⅰ ESCC patients and stage Ⅲ ESCC patients, 2 and 8 circRNAs that were only specifically expressed in paracancerous tissues and had significant differences were screened out respectively, and were only specifically expressed in cancer tissues with significant differences were 11 and 14 respectively. The circRNA-miRNA-mRNA network showed that the cancer tissue-related circRNA-miRNA-mRNA network in stage Ⅰ ESCC patients consisted of 7 circRNA nodes, 10 miRNA nodes and 28 mRNA nodes, and the cancer tissue-related circRNA-miRNA-mRNA network in stage Ⅲ ESCC patients consisted of 7 circRNA nodes, 9 miRNA nodes and 49 mRNA nodes. The most significantly differentially expressed hsa-circ-0060927 and hsa-circ-0109301 in cancer tissues of patients with stage Ⅰ ESCC and stage Ⅲ ESCC were selected for cytological and histological verification. The results showed that the relative expression levels of hsa-circ-0060927 in ESCC cell lines TE1, TE13, KYSE30, KYSE170, and human normal esophageal epithelial cell line HEEC were 7.82±1.96, 12.69±2.68, 12.78±2.74, 7.53±1.75, and 2.43±0.17, respectively, with a statistically significant difference ( F=4.68, P=0.004). The relative expression levels of hsa-circ-0060927 in ESCC cell lines TE1, TE13, KYSE30, and KYSE170 were higher than that in human normal esophageal epithelial cell line HEEC, with statistically significant differences ( P=0.009; P=0.003; P=0.003; P=0.007). The relative expression levels of hsa-circ-0109301 in ESCC cell lines TE1, TE13, KYSE30, KYSE170, and human normal esophageal epithelial cell line HEEC were 5.16±1.32, 6.28±1.57, 4.89±1.13, 8.92±2.12, and 22.56±4.13, respectively, with a statistically significant difference ( F=4.31, P=0.022). The relative expression levels of hsa-circ-0109301 in ESCC cell lines TE1, TE13, KYSE30, and KYSE170 were lower than that in human normal esophageal epithelial cell line HEEC, with statistically significant differences ( P=0.027; P=0.015; P=0.024; P=0.008). The expression level of hsa-circ-0060927 in cancer tissues of 13 early ESCC patients was 12.89±2.67, significantly higher than 5.73±1.18 in paracancerous tissue, and there was a statistically significant difference ( t=15.02, P<0.001) ; the expression level of hsa-circ-0109301 in cancer tissues of 19 patients with advanced ESCC was 7.78±2.17, significantly lower than 16.32±3.15 in paracancerous tissue, and there was a statistically significant difference ( t=9.73, P<0.001). The expression of hsa-circ-0109301 was related to the degree of tumor differentiation in advanced ESCC patients ( P=0.023) . Conclusion:One circRNA (hsa-circ-0060927 and hsa-circ-0109301) with the most significanty differential expression is selected in early and advanced ESCC patients respectively, in which hsa-circ-0060927 is highly expressed in ESCC cancer tissues and hsa-circ-0109301 is lowly expressed in ESCC cancer tissues, and the expression of hsa-circ-0109301 is correlated with the degree of tumor differentiation.

Transcriptomics Provides Novel Insights into the Regulatory Mechanism of IncRNA HIF1 A-AS1 on Vascular Smooth Muscle Cells

ABSTRACT Introduction: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics. Methods: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results. Results: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways. Conclusion: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

Identification of novel variants in retinitis pigmentosa genes by whole-exome sequencing

SUMMARY OBJECTIVE: Retinitis pigmentosa is an inherited degenerative disorder causing severe retinal dystrophy and visual impairment, mainly with onset in the first or second decades. The next-generation sequencing has become an efficient tool to identify disease-causing mutations in retinitis pigmentosa. The aim of this retrospective study was to investigate novel gene variants and evaluate the utility of whole-exome sequencing in patients with retinitis pigmentosa. METHODS: The medical records of 20 patients with retinitis pigmentosa at Eskişehir City Hospital between September 2019 and February 2022 were analyzed retrospectively. Peripheral venous blood was obtained, followed by the extraction of genomic DNAs. The medical and ophthalmic histories were collected, and ophthalmological examinations were performed. Whole-exome sequencing was performed to determine the genetic etiology of the patients. RESULTS: The proportion of genetically solved cases was 75% (15/20) in the patients with retinitis pigmentosa. Molecular genetic testing identified 13 biallelic and 4 monoallelic mutations in known retinitis pigmentosa genes, including 11 novel variants. According to in silico prediction tools, nine variants were predicted as pathogenic or possibly pathogenic. We identified six previously reported mutations to be associated with retinitis pigmentosa. The age of onset of the patients ranged from 3 to 19, with a mean age of onset of 11.6. All patients had a loss of central vision. CONCLUSION: As the first study of the application of whole-exome sequencing among patients with retinitis pigmentosa in a Turkish cohort, our results may contribute to the characterization of the spectrum of variants related to retinitis pigmentosa in the Turkish population. Future population-based studies will enable us to reveal the detailed genetic epidemiology of retinitis pigmentosa.

Molecular and functional study of pediatric patients with Niemann-Pick C in Argentina / Estudio molecular y funcional de pacientes pediátricos con Niemann-Pick C en Argentina

Abstract Niemann-Pick type C (NP-C) is a rare, autosomal recessive disorder. At least 95% of all the cases with this disease are due to mutations in the NPC1 gene. The clinical signs and symptoms of NP-C are classified into visceral, neurological and psychiatric. Our aim is to report the clinical findings, molecular results and filipin staining of 4 patients. The age of onset, expressed as median and range, was 0.2 (0.08-4.0) years and the age of diagnosis was 4.0 (2.5-8.9) years. Neurological and/or visceral manifestations were presented in our patients. Foamy cells in bone marrow biopsy were found in two patients. Through a molecular analysis of NPC1 gene, one non-reported (novel) and 4 previously described mutations were found. The filipin staining showed a positive pattern in all the patients. The diagnostic confirmation of these pediatric patients means a contribution to the casuistry of this disease in Argentina.

Resumen Niemann-Pick tipo C (NP-C) es una enfermedad poco frecuente, con un patrón de herencia au tosómico recesivo. Al menos el 95% de los casos se producen por mutaciones en el gen NPC1. Los signos y síntomas clínicos de NP-C se clasifican en viscerales, neurológicos y psiquiátricos. En este trabajo presentamos los hallazgos clínicos, los resultados moleculares y la tinción con filipina de 4 pacientes con NP-C. La edad de presentación de los primeros síntomas, expresada como mediana y rango, fue de 0.2 años (0.08-4.0) años y la edad del diagnóstico fue 4.0 (2.5-8.9) años. Los pacientes presentaron manifestaciones neurológicas y / o vis cerales. Se encontraron células espumosas en la biopsia de médula ósea en 2 pacientes. El análisis molecular del gen NPC1 encontró 1 variante nueva y 4 previamente publicadas. La tinción de filipina mostró un patrón positivo en todos los pacientes. La confirmación diagnóstica de este grupo de pacientes pediátricos significa un aporte a la casuística de esta enfermedad en Argentina.

Value of detection of cell-free fetal DNA in maternal peripheral blood for Down's syndrome screening / 中国基层医药

Objective:To investigate the value of detection of cell-free fetal DNA in maternal peripheral blood for Down's syndrome screening.Methods:A total of 1667 pregnant women who were at a higher risk of having a baby with Down's syndrome who received Down's syndrome screening in the First People's Hospital of Datong between January 2020 and March 2021 were prospectively analyzed. After detection of cell-free fetal DNA in maternal peripheral blood, pregnant women who were at a higher risk of having a baby with Down's syndrome decided whether to accept amniocentesis for fetal karyotype. Then follow-up was performed for collecting related information. Finally, detection results of cell-free fetal DNA in maternal peripheral blood, fetal karyotype results and pregnancy outcomes were analyzed.Results:The positive predictive value of detecting cell-free fetal DNA in maternal peripheral blood for trisomy 21, trisomy 18, and trisomy 13 and chromosome abnormality were 100.0%, 100.0%, 0.0% and 66.7%, respectively. The sensitivity and total specificity of detecting cell-free fetal DNA in maternal peripheral blood were 100.0% and 99.8%, respectively. The false positive rate of detecting cell-free fetal DNA in maternal peripheral blood for trisomy 13 and chromosome abnormality was 0.12% and 0.06%, respectively.Conclusion:A high degree of coincidence between detection results of cell-free fetal DNA in maternal peripheral blood and fetal karyotype results can be used as a prenatal screening for Down's syndrome. This has certain guiding significance for invasive prenatal diagnosis through amniocentesis-based fetal karyotype analysis.

A novel mutation of SPAST gene in a hereditary spastic paraplegia type 4 family / 中华内科杂志

Objective:To clarify the pathogenicity and further explore the association between genotype and clinical phenotype of this variant, analyzing a novel variation of SPAST gene in hereditary spastic paraplegia (HSP) family from Changzhi city, Shanxi Province.Methods:A family with HSP was tracked and collected in Neurology Department of Heping Hospital Affiliated to Changzhi Medical College in October 2019. Peripheral venous blood of 2 ml was extracted from the proband and 8 other members of the family, genomic DNA was extracted from the blood samples, and the genes of spastic paraplegia were screened by next-generation sequencing (NGS). HGMD, 1000G, OMIM databases and PolyPhen2, SIFT and other software were used for bioinformatics analysis of suspected mutations. Multiplex ligation-dependent probe amplification (MLPA) was used to further screen for total deletions/duplications in patients who remained negative after targeting NGS, and Sanger sequencing was performed to verify the suspected pathogenic mutation sites in the family to determine co-isolation of the mutation sites in the family members. Finally, it is necessary to refer to the latest version of The American College of Medical Genetics and Genomics (ACMG) sequence variation interpretation guidelines to interpret the mutation sites to determine pathogenicity.Results:The HSP family consist 47 members of 4 generations and 10 patients, with onset ages ranging from 2 to 44 years. The proband′s daughter only showed positive bilateral Babbitt signs on physical examination, and the rest of the patients showed spasticity and weakness of lower limbs with varying severity on this basis. Preliminary screening by next-generation sequencing technology showed that the proband had frame-shift variation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) and missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg). Then, Sanger sequencing was used for in-family verification, which showed SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was detected in the affected members include father, brother, son and daughter, and not detected in the unaffected normal members, the proband′s wife, mother, sister and sister-in-law. However, the unaffected of mother detected missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg), while the remaining members did not detect this variation. The results of MLPA showed that no large fragment variation was found.Conclusions:The genetic pattern of the HSP family was autosomal dominant, and the clinical characteristics were consistent with hereditary spastic paraplegia type 4 (SPG4). Co-segregation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was found in the HSP family and was the pathogenicity cause of this SPG4 family, and it was a newly discovered mutation locus.

Analysis of pathogenic genes in two cases of Sj?gren-Larsson syndrome / 中华皮肤科杂志

Objective:To detect pathogenic mutations in 2 patients with Sj?gren-Larsson syndrome.Methods:Clinical data were collected from 2 children with Sj?gren-Larsson syndrome, who were diagnosed and treated in Department of Dermatology, Capital Institute of Pediatrics, and genetic testing was performed to clarify their pathogenic mutations.Results:Both the 2 patients presented with typical ichthyosis-like skin manifestations, accompanied by a certain degree of intellectual disorder and growth, development and motor retardation. A previously reported homozygous mutation c.1157A>G was identified in the ALDH3A2 gene in case 1; compound heterozygous mutations c.1157A>G and c.1309A>T were identified in the ALDH3A2 gene in case 2, which were considered as novel pathogenic mutations.Conclusion:Genetic testing should be performed as early as possible in patients with suspected Sj?gren-Larsson syndrome, in order to facilitate early definite diagnosis.

Molecular genetic and clinical characteristics of MEF2D-BCL9 fusion gene-positive acute B-cell lymphoblastic leukemia / 白血病·淋巴瘤

Objective:To investigate the molecular genetic and clinical characteristics of MEF2D-BCL9 fusion gene-positive acute B-cell lymphoblastic leukemia (B-ALL), and to provide the reference for the diagnosis and treatment of the disease.Methods:The medical record and experimental examination data of a 18-year-old female MEF2D-BCL9 fusion gene-positive B-ALL patient were retrospectively analyzed. The clinical manifestations and biological characteristics of MEF2D-BCL9 fusion gene-positive B-ALL were summarized.Results:This 18-year-old female patient was treated in a local hospital in December 2018 and was diagnosed as B-ALL. She achieved complete remission after chemotherapy and recurred at 6 months after the initial onset, and then she was admitted to Hebei Yanda Ludaopei Hospital in the 9 months after the initial onset.MEF2D-BCL9 fusion gene was detected through RNA-sequencing (RNA-seq) and verified by using polymerase chain reaction and Sanger sequencing. Bone marrow cell morphology was similar to mature B cells with vacuoles but without characteristic chromosome karyotype abnormalities. The patient achieved remission after VLD regimen chemotherapy, chimeric antigen receptor T-cell (CAR-T) therapy and bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT). She has maintained complete remission for 2 years at the last follow-up in February 2022.Conclusions:MEF2D-BCL9 fusion gene-positive B-ALL is characterized with high risk, early relapse and poor prognosis. These patients may benefit from CAR-T and allo-HSCT. It further emphasizes the importance of taking MEF2D-BCL9 fusion gene into the detection or identification by using RNA-seq, particularly for those newly diagnosed B-ALL patients in children and adolescents with specific bone marrow morphology.