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Objective: To explore the serological characteristics and molecular mechanism underlying an individual with A3 phenotype., Methods: A 27-year-old ethnic Han Chinese woman presented at the Fourth Affiliated Hospital of China Medical University on May 12, 2022 was selected as the study subject. ABO blood type was determined with standard serological techniques. The ABO gene was subjected to direct sequencing of PCR products. Exons 6 and 7 of the ABO gene were sequenced using specific primers to determine the haplotypes. Bioinformatic software was used to analyze the structure of the mutant protein., Results: Serological typing of the ABO blood group has suggested a rare A3 phenotype. The proband was found to harbor heterozygous c.261delG, c.467C>T and c.745C>T variants by direct sequencing. Single strand sequencing revealed that she has harbored ABO*A3.07 and ABO*O.01.01 alleles. The ABO*A3.07 allele has contained a c.745C>T (p.R249W) variant on the background of an ABO*A1.02 allele. The p.R249W substitution was predicted to be probably damaging by the PolyPhen2 software. The free energy change (ΔΔG) value predicted it to have a destabilizing effect on the GTA protein. Meanwhile, modeling of the 3D structure has predicted that the p.R249W amino acid substitution may alter the hydrogen bond network of the GTA protein., Conclusion: The p.R249W substitution of the α-1,3-N-acetylgalactosaminyltransferase gene may reduce the antigen expression owing to a great destabilizing effect on the structure and function of the GTA protein.

Objective: To explore the genetic basis of a myopathic patient with pathological characteristics including tubular aggregates and vacuoles., Methods: Next generation sequencing was carried out for the patient, and candidate variant was verified by Sanger sequencing., Results: Genetic testing revealed that the patient has harbored a heterozygous c.730G>C (p.D244H) variant of Calsequestrin 1 (CASQ1) gene. The same variant was not found in his unaffected parents. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was rated as pathogenic (PS1+PM2+PP3)., Conclusion: The novel c.730G>C (p.D244H) variant of the CASQ1 gene probably underlay the myopathy in this patient. Above finding has enriched the mutational spectrum of the CASQ1 gene.

Objective: To analyze the clinical features and genetic basis of a child with Disorder of sex development (DSD)., Methods: A child who was admitted to the Linyi People's Hospital for primary amenorrhoea on July 29, 2019 was selected as the study subject. Clinical data of the child was collected. Chromosomal karyotyping and quantitative real-time PCR were used to detect Y chromosome microdeletions and other chromosomal aberrations. Next-generation sequencing was carried out for the child and her parents. Candidate variant was verified by Sanger sequencing and bioinformatic analysis., Results: The child, a 13-year-old girl, has featured primary amenorrhoea and onset of secondary sex characteristics of males. Ultrasound exam had detected no uterus and definite ovarian structure, but narrow band vaginal hypoecho and curved cavernoid structure. The child was found to have a 46,XY karyotype without an AZF deletion. DNA sequencing revealed that she has harbored a maternally derived c.323delA (p.Q108Rfs*188) variant in the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene, which may result in a truncated protein. The variant was classified as pathogenic (PVS1+PM2_Supporting+PP4) based on the guidelines from the American College of Medical Genetics and Genomics., Conclusion: The NR5A1: c.323delA variant probably underlay the pathogenesis of 46,XY DSD in this child. The discovery of the novel variant has enriched the mutational spectrum of the NR5A1 gene and provided a basis for clinical diagnosis, treatment and prenatal diagnosis.

Objective: To study the serological characteristics of ABO*A2.08 subtype and explore its genetic molecular mechanism., Methods: ABO blood group identification was performed on proband and her family members by routine serological methods. ABO genotyping and sequence analysis were performed by polymerase chain reaction-sequence specific primer (PCR-SSP), and direct sequencing of PCR products from exons 6 and 7 of ABO gene were directly sequenced and analyzed. The effect of gene mutation in A2.08 subtype on structural stability of GTA protein was investigated by homologous protein conserved analysis, 3D molecular modeling and protein stability prediction., Results: The proband's serological test results showed subtype Ax, and ABO genotyping confirmed that the proband's genotype was ABO*A207/08 . Gene sequencing of the proband's father confirmed the characteristic variation of c.539G>C in the 7 th exon of ABO gene, leading to the replacement of polypeptide chain p.Arg180Pro (R180P). 3D protein molecular modeling and analysis suggested that the number of hydrogen bonds of local amino acids in the protein structure was changed after the mutation, and protein stability prediction showed that the mutation had a great influence on the protein structure stability., Conclusion: The mutation of the 7 th exon c.539G>C of ABO gene leads to the substitution of polypeptide chain amino acid, which affects the structural stability of GTA protein and leads to the change of enzyme activity, resulting in the A2.08 phenotype. The mutated gene can be stably inherited.

Objective: To explore the clinical features and genetic basis for a child with 3-methylglutaconic aciduria type VII., Methods: A child who was diagnosed at the Gansu Provincial Maternity and Child Health Care Hospital on August 9, 2019 was selected as the study subject. Clinical data of the child, including urine gas chromatography and mass spectrometry, were collected. The child and her parents were subjected to whole exome sequencing., Results: The child, a female neonate, had presented mainly with intermittent skin cyanosis, convulsions, hypomagnesemia, apnea, neutropenia after birth. Her urine 3-methylpentenedioic acid has increased to 17.53 μmol/L. DNA sequencing revealed that she has harbored compound heterozygous variants of the CLPB gene, namely c.1016delT (p.L339Rfs*5) and c.1087A>G (p.R363G), which were respectively inherited from her mother and father. Both variants were unreported previously. Based on the standards from the American College of Medical Genetics and Genomics (ACMG), the variants were respectively predicted to be pathogenic and likely pathogenic., Conclusion: The child was diagnosed with 3-methylglutenedioic aciduria type VII. Discovery of the c.1016delT and c.1087A>G variants has enriched the mutational spectrum of the CLPB gene.

Dracaena marginata is a widely cultivated horticultural plant in the world, which has high ornamental and medicinal value. In this study, the whole genome of leaves from D . marginata was sequenced by Illumina HiSeq 4000 platform. The chloroplast genome were assembled for functional annotation, sequence characteristics and phylogenetic analysis. The results showed that the chloroplast genome of D . marginata composed of four regions with a size of 154 926 bp, which was the smallest chloroplast genome reported for Dracaena species to date. A total of 132 genes were identified, including 86 coding genes, 38 tRNA genes and 8 rRNA genes. Codon bias analysis found that the codon usage bias was weak and there was a bias for using A/U base endings. 46 simple sequence repeat and 54 repeats loci were detected in the chloroplast genome, with the maximum detection rate in the large single copy region and inverted repeat region, respectively. The inverted repeats boundaries of D . marginata and Dracaena were highly conserved, whereas gene location differences occurred. Phylogenetic analysis revealed that D . serrulata and D . cinnabari form a monophyletic clade, which was the closest relationship and conformed to the morphological classification characteristics. The analysis of the chloroplast genome of D . marginata provides important data basis for species identification, genetic diversity and chloroplast genome engineering of Dracaena .

Objectives: To study the association of ventricular septal defect (VSD) with rare variations in the promoter region of HAND2 gene, as well as related molecular mechanisms., Methods: Blood samples were collected from 349 children with VSD and 345 healthy controls. The target fragments were amplified by polymerase chain reaction and sequenced to identify the rare variation sites in the promoter region of the HAND2 gene. Dual-luciferase reporter assay was used to perform a functional analysis of the variation sites. Electrophoretic mobility shift assay (EMSA) was used to investigate related molecular mechanisms. TRANSFAC and JASPAR databases were used to predict transcription factors., Results: Sequencing revealed that three variation sites (g.173530852A>G, g.173531173A>G, and g.173531213C>G) were only observed in the promoter region of the HAND2 gene in 10 children with VSD, among whom 4 children had only one variation site. The dual-luciferase reporter assay revealed that g.173531213C>G reduced the transcriptional activity of the HAND2 gene promoter. EMSA and transcription factor prediction revealed that g.173531213C>G created a binding site for transcription factor., Conclusions: The rare variation, g.173531213C>G, in the promoter region of the HAND2 gene participates in the development and progression of VSD possibly by affecting the binding of transcription factors.

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Objective: To investigate the effect of ABO gene α-1,3-D galactosyl transferase mutation on B antigen expression and its molecular mechanism., Methods: The proband and their family members were identified by routine serological methods, and ABO genotyping and sequence analysis were performed by polymerase chain reaction-sequence specificity (PCR-SSP) and direct sequencing of PCR products from exon 1-7 of ABO gene. The 3D structural simulation of mutant proteins was performed by bioinformatics software. The effect of gene mutation on protein structural stability was analyzed., Results: The proband and his family members were subtype B. ABO genotyping indicated that the proband's genotype was Bw12/O. Gene sequencing results confirmed the presence of ABO*BW.12 characteristic variation c.278C>T in the 6th exon of allele B, leading to the replacement of polypeptide chain p.Pro93Leu. The 3D structure simulation analysis of the protein showed that the hydrogen bonds and water molecules connected to the protein changed after amino acid substitution. The family investigation found that the grandfather, father, uncle and brother of the proband all carried the same ABO*BW.12 allele., Conclusion: The mutation of the 6th exon c.278C>T of ABO gene led to the substitution of polypeptide chain amino acids, which affected the stability of α-1,3-D galactosyl transferase protein, resulting in the change of enzyme activity, and the Bw.12 phenotype, which can be stably inherited.

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes Lgu Ⅰ and Bbv CⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of Lgu Ⅰ and Bbv CⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB- welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Traditional bulk RNA sequencing assesses the average expression level of genes in tissues rather than the differences in cellular responses. Accordingly, it is hard to differentiate sensitive responding cells, leading to inaccurate identification of toxicity pathways. Single-cell RNA sequencing (scRNA-seq) isolated single cells from tissue and subjected them to cell subtypes-specific transcriptome analysis. This technique in toxicological studies realizes the heterogeneous cellular responses in the tissue microenvironment upon chemical exposure. Thus it helps to identify sensitive responding cells and key molecular events, providing a powerful tool and a new perspective for exploring the mechanisms of toxicity and the modes of action. This review summarizes the development, principle, method, application and limitations of scRNA-seq in mechanistic toxicological researches, and discusses the prospect of multi-directional applications.

Objective: To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1 mut ) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype., Methods: The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1 mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum., Results: Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1 mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1 mut /FLT3-ITD + and 4.0 (range 2－14) for NPM1 mut /FLT3-ITD - cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1 mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1 mut /FLT3-ITD - patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1 mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly., Conclusion: Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1 mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

The emergence of single-cell sequencing technology enables people to observe cells with unprecedented precision. However, it is difficult to capture the information on all cells and genes in one single-cell RNA sequencing (scRNA-seq) experiment. Single-cell data of a single modality cannot explain cell state and system changes in detail. The integrative analysis of single-cell data aims to address these two types of problems. Integrating multiple scRNA-seq data can collect complete cell types and provide a powerful boost for the construction of cell atlases. Integrating single-cell multimodal data can be used to study the causal relationship and gene regulation mechanism across modalities. The development and application of data integration methods helps fully explore the richness and relevance of single-cell data and discover meaningful biological changes. Based on this, this article reviews the basic principles, methods and applications of multiple scRNA-seq data integration and single-cell multimodal data integration. Moreover, the advantages and disadvantages of existing methods are discussed. Finally, the future development is prospected.

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.

Objective: To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni., Methods: Total RNA was extracted from S. mansoni , and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni . Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted., Results: The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 10 6 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively., Conclusions: A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

Objective: To verify a rare allele of human leukocyte antigen (HLA) and analyze its inheritance and 3D molecular structure., Methods: PCR-sequence-based typing, PCR-single strand oligonucleotide polymorphism and single allele-specific sequencing were carried out to characterize the rare HLA-C allele and its transmission in the family. Its protein structure was modeled by using SWISS-MODEL, Phyre2 and FATCAT software., Results: Analysis indicated that the rare allele (HLA-C*08:84) has transmitted from the proband's mother and has differed from HLA-C*08:01 by a single base (g.512G>C), resulting in substitution of an amino acid (p.Trp147Ser). Modeling of the 3D structure of the encoded protein indicated that the amino acid residue variation is located at the alpha 2 helix, which participates the formation of pocket F. Modeling of the structures of C*08:84, C*08:01, C*08:02, C*08:03 and C*08:22 has suggested significant variation in the peptide binding regions of the backbone, with root mean square errors being 1.70 nm, 1.79 nm, 0.71 nm and 1.70 nm, respectively., Conclusion: A rare HLA-C*08:84 allele has been identified, and its clinical significance has been analyzed.

Objective: To delineate the characteristics of a novel HLA-DQB1 allele identified during routine HLA matching in a leukemia family., Methods: The mother and brother of the patient were subjected to PCR sequence-specific oligonucleotide probe (SSOP), PCR sequence-based typ1ing (SBT), as well as next-generation sequencing (NGS)., Results: PCR-SBT revealed that the patient's mother and brother's HLA-DQB1 sequences did not fully match with any known allele combination. NGS revealed that the novel allele has differed from the closest matched DQB1*03:02 with a T>G substitution at position 233 in exon 2, which resulted in substitution of Valine at codon 46 by Glycine. Pedigree analysis confirmed that the novel HLA-DQB1 allele was inherited from his mother., Conclusion: A novel HLA-DQB1 allele has been identified through next generation sequencing and was officially named as HLA-DQB1*03:362 by the World Health Organization HLA Factor Nomenclature Committee.

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.

Objective: To explore the molecular basis for an individual suspected as AwB subtype through DNA sequencing., Methods: ABO serology was carried out with the standard tube method. To identify the ABO gene haplotype, the amplicons of exon 7 were cloned and sequenced., Results: Serological results showed that the forward typing was AwB and the reverse typing was B. Sequencing analysis revealed that the sample has contained an O01 allele in addition with c.297A>G, c.657C>T, c.796C>A, c.803G>C, c.930G>A variants as compared with the A101 allele., Conclusion: Through sequencing analysis, the sample with an AwB subtype by serological testing was identified as a novel B(A) phenotype, which was unreported previously.

Streptomyces aureofaciens DM-1 is a high-yielding 6-demethylchlortetracycline producer. The genome sequencing of DM-1 reveals a linear chromosome containing 6 824 334 bps nucleotides with GC content of 72.6%. In this genome, a total of 6 431 open reading frames were predicted by using glimmer 3.02, Genemark and Z-Curve softwares. Twenty-eight secondary metabolite biosynthetic gene clusters were uncovered by using AntiSMASH gene prediction software, including the complete 6-demethylchlortetracycline biosynthetic gene cluster. A frame-shift mutation in methyltransferase coding region was detected, which may result in the demethylation of chlortetracycline. The complete genome sequence of S. aureofaciens DM-1 provides basic information for functional genomics studies and selection of high-yielding strains for 6-demethylchlortetracycline.

Objective: To characterize Torso-like ( tsl ) gene and investigate its expression characteristics in Anopheles dirus , so as to provide a theoretical basis for subsequent functional studies of the tsl gene., Methods: According to the coding sequences of Drosophila melanogaster and An. gambiae tsl genes, the complete genome of An. dirus was retrieved and the An. dirus tsl gene was characterized. Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays. The physicochemical properties, signal peptide, transmembrane structure, secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools, and a phylogenetic analysis was performed. In addition, the specific expression of the tls gene was detected in various tissues of An. dirus using a quantitative real-time PCR assay., Results: The An. dirus tsl gene was 16 751 bp in length with a CDS region of 1 134 bp, encoding 377 amino acids, and the encoded TSL protein was a stably hydrophilic protein. The TSL protein was predicted to be a secretory protein that was located in extra-membrane regions containing signal peptides. The secondary structure of the TSL protein contained α-helix (51.72%), extended strand (12.20%), β-bridge (4.78%) and random coil (31.30%) in the secondary structure, and a 3D homology model was generated using 5cj9.1.A as a template. Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An. dirus and An. farauti . In addition, quantitative real-time PCR assay detected the tsl gene expression in the head, chest, abdomen and foot of An. dirus , with the highest expression in the head and low expression in the foot., Conclusions: The tsl gene is characterized in An. dirus at a genomic level, and the prediction of the TSL protein structure and the elucidation of the tissue-specific tsl gene expression in An. dirus provide a basis for the further studies on the gene functions.

Objective: To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4)., Methods: Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing., Results: DNA sequencing suggested that the proband has carried a heterozygous c.1196C>G variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic., Conclusion: The c.1196C>G variant of the SPAST gene probably underlay the HSP4 in this pedigree.

Objective: To obtain the transcriptome data of Tyrophagus putrescentiae , so as to provide insights into the subsequent functional studies., Methods: The mixture of male and female T. putrescentiae was sequenced using the Illumina HiSeqTM 2000 high-throughput sequencing platform. Unigenes were obtained after assembling the sequencing data using the Trinity software and compared with the protein sequences in the RefSeq non-redundant protein sequence (NR) database, nucleotide sequence (NT) database, Swiss-Prot database, Kyoto encyclopedia of genes and genomes (KEGG) database and clusters of orthologous groups (COG) database, and the function of the Unigenes was annotated. In addition, the coding DNA sequences (CDS) were predicted through alignment of the Unigenes in NR and Swiss-Prot protein databases. The SSR loci were identified by analysis of the Unigenes in T. putrescentiae with the MISA software, and the SNPs were detected using the SOAPsnp technique., Results: A total of 4.67 GB high-quality data were obtained from raw sequencing data. A total of 51 271 Unigenes were obtained after assembling the sequencing data, with a total length of 41 848 995 nucleotide (nt) and a mean length of 816 nt. A total of 29 053 annotated Unigenes were obtained following comparisons with the public protein databases, and 27 443 CDS were predicted. In addition, there were 23 092 SSR loci and 148 027 SNPs identified., Conclusions: The database of T. putrescentiae transcriptome is created by sequencing, and a large number of T. putrescentiae transcripts are obtained, which provides a basis for the subsequent functional studies of allergy-related genes.

Objective: To analyze serological and molecular characteristics of a case with Bw11 subtype., Methods: The ABO antigen and antibody in serum were respectively detected with the classical tube method, microcolumn gel method, and absorption and diffusion method. The ABO genotype was determined with PCR using sequence-specific primers (PCR-SSP). Exons 1-7 of the ABO gene were analyzed by Sanger sequencing. Haplotype analysis was carried out for exons harboring variants., Results: Forward and reverse typing with the microcolumn gel method has suggested type O, while forward and reverse typing with the classical tube method yielded inconsistent results. Absorption and diffusion test confirmed presence of B antigen. Antibody screening excluded presence of alloantibodies. The result of PCR-SSP suggested a B/O1 genotype.A 695T>C variant was identified in exon 7 as compared with the B101/O01 allele, which resulted in conversion of Leucine to Proline at position 232, and was confirmed as Bw11/O1 heterozygote., Conclusion: The nt695T>C variant probably underlay the weakening of B antigenin the individual. There may be strong anti-B antibodies in Bw11 subtypes. Human-derived and certain monoclonal reagents may not detect Bw11 subtypes which is easy to be misjudged as type O. Application of molecular methods can identify ABO subtypes with accuracy.

Gene panel and whole exome sequencing are now commonly used to detect Mendelian disease, but the current molecular diagnostic rate of DNA sequencing is only 35%-50%. In recent years, RNA sequencing emerges as a promising diagnostic method. It can detect new pathogenic mutations, and analyze allele-specific expression. This will be helpful to understand the relationship between disease genotype and phenotype, and can complement genome sequencing in order to expand the traditional genomic diagnostic methods of Mendelian disease. RNA sequencing is expected to become a routine tool for diagnosing Mendelian diseases. This article reviews the application of RNA sequencing in the clinical diagnosis of Mendelian disease.

Objective: To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1)., Methods: In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene., Results: Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly., Conclusion: A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.

Objective: To report on a novel KIR3DL3 allele identified in a southern Han Chinese individual., Methods: Peripheral blood sample was collected from a voluntary blood donor with inconclusive result by KIR3DL3 sequence-based typing (SBT). Total mRNA was extracted and subjected to reverse transcription to obtain KIR3DL3 cDNA, which was then amplified by PCR with a pair of KIR3DL3-specific primers. The product was subjected to cDNA cloning and sequencing., Results: cDNA cloning and sequencing have identified a wide-type KIR3DL3*00802 allele and a novel KIR3DL3*064 allele. The latter differed from KIR3DL3*00601 by a missense variant at codon 374[c.1184 C>T (p.Thr374Ile)] in exon 9. The novel KIR3DL3 allele has been officially assigned by the KIR subcommittee of World Health Organization Nomenclature Committee for factors of HLA system., Conclusion: cDNA cloning and sequencing may be used to distinguish inconclusive results in KIR3DL3 SBT in order to identify novel KIR alleles.

This article reported the clinical characteristics and SRD5A2 gene mutation pattern of a child with steroid 5-α reductase type 2 deficiency. The 2-month-old boy showed hypospadias and short penis shortly after birth. DNA was extracted from the peripheral blood of the child and his parents. The endocrine disease-related genes were captured and sequenced by high-throughput sequencing technology, and the family DNA samples were verified by Sanger sequencing. The results showed that c.680G>A(p.R227Q) and c.608G>A(p.G203D) compound heterozygous mutations existed in the SRD5A2 gene of the child. The c.680G>A mutation inherited from his father, which was a known pathogenic mutation. The c.608G>A mutation originated from his mother, which was a novel mutation discovered in this study. These results provide molecular evidence for the etiological diagnosis of the child and genetic counseling for the family, as well as extend the mutation spectrum of SRD5A2 gene.

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Sequencing technology has been greatly improved in terms of throughput and cost. The single-molecule nanopore DNA sequencing, one of the major branches of the third-generation sequencing technology, has made great contributions in the fields of medicine and life sciences due to its advantages of ultra-long reading length, real-time detection and direct detection of base methylation modification, etc. This article briefly describes the principle of nanopore sequencing technology, and discusses its application in clinical, animal, plant, bacterial and virus fields and its future development direction.

Objective: To analyze the amino acid polymorphism of truncated Staphylococcal enterotoxin-like toxin X (tSElX), and to evaluate its related emetic activities. Methods: Sequence of tselx was compared with both the genome sequence of 145 CC398 strains completed in our research group and the NCBI database. Primers were designed to amplify the target gene of tselx , and the fragment was recombined into pMD18-T vector and sequenced. PCR product was digested with BamH Ⅰ and EcoR Ⅰ, and constructed into plasmid pGEX-6P-1 and pET-28a (+). After recombinant plasmid was identified, the protein expression was induced by IPTG. Proteins expressed in the form of inclusion bodies were denatured and renatured, then purified by affinity chromatography and ultrafiltration. Purified tSElX protein was then fed to common marmosets with the dose of 250 μg/kg to observe the vomiting reaction. Results: gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0 tselx gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0 % , while the homology with the four American strains were 97.8 % (1) and 98.9 % (3), respectively. Through two sets of expression systems and different induction conditions, tSElX was expressed in the form of inclusion bodies. The high purity soluble recombinant tSElX was thus obtained by denaturated and renaturated processes. At the dose of 250 μg/kg, tSElX protein did not cause vomiting in common marmosets. Conclusions: Results of this study showed that the amino acid sequence of tSElX was highly conserved and was universally present in a particular clone group. We obtained soluble recombinant tSElX protein with high purity. We also noticed that tSElX did not have the animal emetic activity at a dose of 250 μg/kg.

Objective: To explore the clinical feature, genetic variant and clinical outcome of patients with cblA-type methylmalonic acidemia (MMA)., Methods: Clinical manifestations, therapeutic schedule and prognosis of 12 patients with cblA type MMA were analyzed. MMAA gene variants were analyzed for all patients and their parents., Results: Vomiting, dyspnea and drowsiness were the major clinical features of cblA-type MMA. Eleven patients were vitamin B12-responsive. After treatment, the blood level of propionylcarnitine, ratio of propionylcarnitine/acetylcarnitine, urine level of methylmalonic acid and methylcitric acid have decreased significantly (P< 0.05). Follow-up study showed that 8 patients (66.7%) had normal development, while the rest (33.3%) remained to have various level of mental or movement delay. Fourteen MMAA gene variants were detected, with c.365T>C (p.L122P) being the most common (29.2%). Six novel variants, including c.54delA (p.A19Hfs*43), c.275G>A (p.G92V), c.456delT (p.G153Vfs*8), c.667dupA (p.T223Nfs*4), c.1114C>T (p.Q372X) and c.1137&#95;1138delCA (p.F379Lfs*27) were found., Conclusion: The main clinical manifestations of patients with cblA-type of MMA include vomiting, dyspnea and drowsiness. Most patients are vitamin B12-responsive. c.365T>C is a potential hot spot variant of MMAA gene in China.

Objective: To analyze the genetic characteristics of a five generations pedigree with homozygous familial hypercholesterolemia (HoFH). Methods: Prospective study. Twenty family members included a proband diagnosed as familial hyperlipidemia at the cardiology Department of Xi'an Children's Hospital in October 2018 were research object. Clinical data were collected. Genome DNAs were extracted. Whole exons sequencing was performed on the proband using target capture next generation sequencing. Candidate gene mutation sites identified by bioinformatics were verified by Sanger sequencing in the family members. The genotype-phenotype correlation of the pedigree was analyzed between heterozygous mutation carriers and non-carriers. Results: The proband was a 7-years and 10-month-old boy. He was born with a roundgreen bean size yellow skin protuberance in the skin of the coccyx. Since the age of 3-4 years old, xanthoma-like lesions with a diameter of 0.5-1.5 cm gradually appeared in the skin of bilateral elbow joints, knee joints and Achilles tendon. The height, weight and intellectual development of the child were the same as those of normal children at the same age. No similar xanthoma-like lesion was found in the other family members. The proband's total cholesterol (TC) reached 18.16-21.24 mmol/L, and his low density lipoproteincholesterol (LDL-C) was 14.08-15.51 mmol/L. Carotid ultrasonography showed diffuse sclerotic plaques in bilateral carotid and vertebral arteries, and color Doppler echocardiography revealed aortic valve thickening and calcification. Gene testing identified that the proband carried a homozygous mutation C. 418G>A (p. E140K) in LDLR gene inherited from his parents who had a consanguineous marriage and carried a heterozygous mutation of LDLR-E140K, respectively.The TC, LDL-C and apolipoproteinB (ApoB) of LDLR-E140K gene heterozygous carriers ((8.40±0.13), (6.79±0.01) and (1.95±0.05) mmol/L, respectively) were significantly higher than those of non-carriers ((4.59±0.28), (3.35±0.39) and (0.86±0.10) mmol/L, t= 7.269, 4.595, 6.311, respectively, P< 0.05). Conclusions: LDLR-E140K gene homozygous mutation is first reported to be associated with most severe phenotype HoFH. The genotype-phenotype analysis of the pedigree shows that the clinical phenotype of the proband with homozygous mutation is the most serious, and all the heterozygous mutation carriers present with hypercholesterolemia phenotype. The investigation confirms that LDLR-E140K is the pathogenic variation of familial hyperlipidemia.

Objective: To identify a novel human leukocyte antigen (HLA) B allele in a Chinese Han individual and construct its three-dimensional structure., Methods: The initial HLA genotyping was performed by PCR-sequence-based typing (PCR-SBT). The ambiguous allele was confirmed with single-strand DNA sequencing. The DNA sequence was analyzed to identify the difference between the novel allele and its closest matching allele. Finally, the three-dimensional molecular structure of the novel allele was constructed using a Swiss-Model., Results: One allele of the subject at the HLA-B locus was B*44:03:01, whilst the other was a novel allele which differed from the closest matching allele B*51:01:01:01 by nucleotide (nt) 329 A to C in exon 2, resulting in an amino acid change at codon 86 (p.Asn86Thr)., Conclusion: A novel HLA-B allele has been identified and officially named as HLA-B*51:159 by the WHO Nomenclature Committee for Factors of the HLA System. The three-dimensional structure of B*51:159 was simulated.

Objective: To identify pathogenic variations of EXT1 and EXT2 genes in two Chinese pedigrees affected with hereditary multiple exostosis (HME)., Methods: Genomic DNA was extracted from peripheral blood samples using a phenol-chloroform method. PCR and Sanger sequencing was conducted to amplify the exons and the flanking intronic regions of the EXT1 and EXT2 genes., Results: DNA sequencing has revealed a heterozygous missense variation c.812A>G (p.Tyr271Cys) in the exon 1 of EXT1 in pedigree 1, and a heterozygous frameshift variation c.1431dup (p.Ser478Leufs*43) in the exon 6 of EXT1 in the proband from pedigree 2. Both variations have co-segregated with the disease phenotype, which was also consistent with previous report., Conclusion: Two heterozygous pathogenic variations underlying HME have been identified. The result has facilitated genetic counseling and prenatal diagnosis for the affected pedigrees.

Background: DNA polymerase β is one of the key enzymes for DNA repair and it was reported that about 30 percent of different types of cancers carried mutations in its coding gene Polb. However, it is still controversial whether it is true or false because of the small sample size in these studies. In current study, we performed genetic screening of promoter and coding regions of Polb gene in 69 Chinese lung cancer patients using Sanger sequencing method, so as to elucidate real mutation frequency of Polb mutations in Chinese Han population., Methods: Salting out extraction method was used to get the genome DNAs from tumor and normal matched tissues of 69 lung cancer patients. The promoter and 14 coding regions of Polb gene were then amplified using these DNAs as the template. After purification, amplicons were sequenced and aligned to the wild type Polb gene in NCBI database, in order to find out the mutated sites of Polb gene in Chinese lung cancer patients., Results: In this study, we totally found only 5 mutated sites in Polb gene. In detail, 3 mutations (-196G>T, -188&#95;-187insCGCCC, -168C>A) were located in the promoter region; 2 mutations (587C>G, 612A>T) were found in coding regions. Specially, mutations of -188&#95;-187insCGCCC and 587C>G (resulting to the amino acid substitution of Thr to Ser at position 196) had never been reported by other groups before. However, all these 5 mutated sites could be detected in both tumor and matched normal tissues, which inferred that they are not lung tumor specific mutations., Conclusions: No lung tumor specific mutations of Polb gene could be found in Chinese lung cancer patients and Polb gene mutation might not be a molecular marker for Chinese lung cancer patients.

Gene structure and expression regulation mechanism are the research hotspots and focus of modern life sciences. The lac operon is a cluster of genes through which Escherichia coli catabolizes lactose. It was first proposed by F. Jacob and J. Monod, who were also awarded the Nobel Prize in Physiology or Medicine in 1965 for their contributions. Thereafter, the lac operon has become the classic teaching case of the gene regulation mechanism in microbiology, genetics and molecular biology, and been highly valued by teachers and students alike. Although the conclusion is easy to follow and memorize, its rich connotation and esoteric reasoning has rendered it difficult to understand, neither is it easy for teachers to fully exploit the advantages of this teaching case. Therefore it is necessary to have an in-depth understanding of the genetic structure and working principle of the lac operon, especially the scientific background and thinking process through which scientists revealed these mysteries. In this paper, the historical discovery and analysis process of the E. coli lac operon was reviewed by following their footprints, listening to their analysis of experimental results. Based on the DNA sequences, the reasons for several unusual phenomena of lac operon expression were also discussed to exemplify the teaching value of the classic cases in genetics and molecular biology.

Objective: To identify mutation of the PAX6 gene in a patient with congenital aniridia., Methods: DNA was extracted from peripheral blood sample of the patient and analyzed by direct PCR-Sanger sequencing., Results: The proband was found to harbor a heterozygous c.239T>A (p.Ile80Asn) mutation of the PAX6 gene. The same mutation was not found in his parents and 150 healthy controls., Conclusion: A novel mutation of the PAX6 gene has been identified in a sporadic case with congenital aniridia.

Objective: To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP)., Methods: Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated., Results: The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1G>C, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic., Conclusion: The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

Objective: To detect potential variant of AR gene in an infant with complete androgen insensitivity syndrome., Methods: The coding regions and splicing sites of the AR gene were subjected to PCR amplification and direct DNA sequencing. Fluorescence quantitative PCR was also used to detect copy number alterations of exons 2 to 8 of the AR gene., Results: Deletion of exons 2 to 8 was detected in the proband, and the results were verified among the family members., Conclusion: Hemizygotic deletion of exons 2 to 8 of the AR gene probably underlies the complete androgen insensitivity syndrome in this infant.

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.

Objective: To explore the potential pathogenetic mutations of primary hypereosinophilia（HEN）by sequencing FGFR1 FLT3, MPL and JAK2 genes, and to clarify their effect on clinical manifestation and prognosis of HEN patients., Methods: The direct DNA sequencing was employed to detect the gene mutations of FGFR1, FLT3, MPL and JAK2 in HEN patients., Results: One deletion mutation (2654&#95;2753del) within tyrosine kinase domain of FLT3 gene was found in a patient suffered from severe symptoms and ended with dismal outcome, which induced a premature stop codon (G885fsX888). For FGFR1, a new variation described as 1014&#95;1019del AACAGT for nucleotide change was found in 19 cases, resulting in T339&#95;V340del at the protein level., Conclusion: The deletion of 6 bases in the FGFR1 gene (1014&#95;1019del AACAGT) is first reported as non-synonymous SNP (nsSNP) site in the patients with primary hypereosinophilia. Deletion mutations in the FLT3 gene may be related with malignant clinical features and poor prognosis.

Objective: To explore the clinical and genetic features of a child with primary ciliary dyskinesia., Methods: Genomic DNA of the child and her parents was extracted and subjected to targeted gene capture and next generation sequencing. Suspected mutation was verified by Sanger sequencing, with its nature and impact predicted by Bioinformatic analysis., Results: Clinical manifestations of the child mainly included severe pneumonia, bronchiectasia, nasosinusitis and pneumothorax. DNA sequencing showed that she has carried compound heterozygous mutations of the CCNO gene, namely c.848T>C (p.L283P) and c.262&#95;263 insGGCCCGGCCC (p.Q88Rfs*51), which were respectively inherited from her mother and father., Conclusion: The child was diagnosed with primary ciliary dyskinesia caused by the compound heterozygous mutations of the CCNO gene.

Objective: To explore the clinical and genetic features of an infant with citrin deficiency (CD)., Methods: Clinical data of the patient was collected and analyzed. Genomic DNA was extracted from peripheral blood samples collected from the patient and her parents. Targeted exome sequencing was performed to explore the genetic cause, and Sanger sequencing was used to confirm the detected variants. SLC25A13 mRNA was extracted from peripheral blood lymphocytes of the infant. The effect of novel mutation of SLC25A13 was analyzed by reverse transcription-PCR, cDNA cloning and Sanger sequencing., Results: The SLC25A13 genotype of the patient was determined as c.845&#95;c.848+1delG/c.1841+3&#95;1841+4delAA, with the latter having not been reported. The mutation has affected the splicing of the SLC25A13 mRNA, giving rise to an aberrant transcript [r.1841&#95;1842ins1841+1&#95;1841+67; 1841+3&#95;c.1841+4del]., Conclusion: A novel SLC25A13 mutation c.1841+3&#95;1841+4delAA and the resultant abnormal splicing variant were discovered by combined DNA sequencing and cDNA cloning. The finding has enabled definite diagnosis of CD and enriched the spectrum of SLC25A13 mutations.

Objective: To explore serological and molecular characteristics of a family trio with weak B phenotype., Methods: ABO blood type of the family was determined with a serological method. Genotypes of the ABO gene were determined with PCR-sequence specific primer (PCR-SSP). Sequences of exons 6 and 7 of the ABO gene were analyzed by PCR sequence-based testing., Results: Serologically, the proband and her father were determined as B subtype (Bw), while her mother was of O group. The genotypes of the proband and her father were Bw12/O, while her mother was O01/O01. Sequencing of exons 6 and 7 of the ABO gene confirmed that the proband and her father were Bw12/O01. Compared with B101/O01, the Bw12 subtype carried a missense mutation (278C>T) in exon 6 of the ABO gene., Conclusion: The 278C>T mutation probably underlies the Bw phenotype and can be transmitted stably.

Objective: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS)., Methods: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology., Results: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing., Conclusion: The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.

Integrins are cell adhesion receptors, which consists of several transmembrane glycoproteins. They are widely distributed on the cell surface and involved in signal transduction pathways. As a heterodimer, each integrin is composed of one α subunit and one β subunit. Integrins are mainly expressed on lepidopteran hemocytes and involved in cell immune response. The full-length cDNA sequence of BmIntegrin β2 was obtained by PCR and RACE, including 2 434 bp. BmIntegrin β2 was predicted to be a transmembrane protein. The BmIntegrin β2 expression profile was detected by qRT-PCR at L4D3 or L5D3 larval stage, and it was highly expressed in hemocyte and hematopoietic organ. Anti-BmIntegrin β2 polyclonal antibody was generated following prokaryotic expression, protein purification and animal immunization, which is highly specific and effective for recognizing BmIntegrin β2 protein through Western blotting. The results of plasmatocytes adhesion experiment showed that BmIntegrin β2 plays an important role on the adhesion and spreading of plasmatocytes to foreign surfaces. This study provides a foundation for further research of the biological function of BmIntegrin β2 gene.

Objective: To explore the clinical and genetic characteristics of a family affected by genetic cholestasis., Methods: Clinical data of the patient was collected. Targeted exome sequencing was carried out to detect the pathogenic mutations. The results were confirmed by Sanger sequencing., Results: The patient, a 5-year-old boy, presented with severe cholestatic cirrhosis. Genetic analysis revealed that he has carried compound heterozygous mutations c.1006-2A>G and c.3580C>T (p.R1194X) of the ABCB4 gene, which were inherited from his father and mother, respectively. By structural prediction, the mutation c.3580C>T can give rise to a truncated multi-drug resistance protein 3 (MDR3)., Conclusion: The patient was diagnosed with progressive familial intrahepatic cholestasis type 3 (PFIC-3) based on clinical and molecular findings. Detection of novel mutations of the ABCB4 gene has provided valuable clues for the diagnosis and genetic counseling.

Objective: To study the effect of a 389T>C variant in the α-1,3-N-acetylgalactosaminyltransferase gene on the expression of A antigen by molecular genetic analysis of an individual with Ax subtype of ABO variant., Methods: Serological assays were carried out to identify the ABO blood group of the proband. Exons 6 and 7 of the ABO gene were amplified with PCR and directly sequenced. Following identification of the heterozygote site, specific primers were used to determine the haplotype of the ABO gene., Results: Weak A antigen was detected on red blood cells from the proband. And anti-A and anti-B antibodies were detected in the serum. DNA sequencing has identified heterozygous 261delG and 389T/C variants in exons 6 and 7. By haplotype analysis, two alleles Ax22 and O01 were found. Compared with that of A101, the Ax22 allele had a nucleotide change (T to C) at position 389, which resulted in an amino acid substitution (L130P)., Conclusion: T>C at nt389 of the α-1,3-N-acetylgalactosaminyl transferase gene can result in significant reduction of A antigen activity.