Analysis of Correlation Between Coronary Heart Disease and Genetic Polymorphism Detected by Gold Magnetic Nanoparticles Chromatography.

It aimed to explore the correlation of Glu504Lys locus mutation of aldehyde dehydrogenase-2 (ALDH2) with coronary heart disease (CHD) based on gold magnetic nanoparticles (GMNPs) chromatography and amplification refractory mutation system-PCR (ARMS-PCR). 120 CHD patients admitted to the cardiovascular Department of Wenling First People's Hospital affiliated to Wenzhou Medical University from December 2020 to December 2021 were selected as Case group and 80 non-CHD patients admitted during the same period were selected as Ctrl group. The venous blood and indexes of Total Cholesterol (TC), Triglyceride (TG), Low Density Lipoprotein Cholesterol (LDL-C), High Density Lipoprotein Cholesterol (HDL-C), and Fasting Blood Glucose (FBS) were collected. The ARMS-PCR GMNPs chromatography based on ARMS-PCR and immunochromatography assay was adopted to detect gene polymorphism of ALDH2. Correlation between ALDH2 gene polymorphism and risk factors of CHD was analyzed via logistic regression. In contrast to Ctrl group, the genotypes of GG, GA, and AA in Case group were evidently different (P < 0.05), and the frequency of A allelic gene was obviously increased (P < 0.05). Under the dominant model, frequency of GA + AA genotype in Case group was remarkably higher in contrast to Ctrl group (P < 0.05). Under the recessive model, there was no obvious difference in genotype frequency between two groups. In contrast to Ctrl group, TC, LDL-C, and FBS in Case group were notably increased (P < 0.05), while HDL-C was notably decreased (P < 0.05). The distribution frequency of abnormal LDL-C, HDL-C, and FBS in Case group was notably higher in contrast to Ctrl group (P < 0.05). LDL-C and FBS had no obvious effect on the genotypes and frequency distribution of alleles in CHD patients. However, the frequency distribution of genotypes of GA and AA and A allelic gene in patients with abnormal HDL-C was notably lower in contrast to those with normal HDL-C (P < 0.05). Logistic regression analysis showed that abnormal HDC-C with A allelic gene were independent risk factors for CHD (P = 0.001, OR = 1.934). The gene polymorphism of Glu504Lys locus of ALDH2 was closely related to the pathogenesis of CHD, A allelic gene may be a susceptibility gene for CHD, and patients with abnormal HDC-C and carried A allelic gene had relatively higher incidence of CHD.

Identification of a homozygous BBS7 frameshift mutation in two (related) Chinese Miao families with Bardet-Biedl Syndrome.

BACKGROUND: Bardet-Biedl Syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with a wide spectrum of clinical features. To date, mutations in 21 different genes (BBS1-21) have been identified as causing isolated or complex BBS phenotypes. In this report, we present three Chinese Miao ethnic patients who were diagnosed with BBS on the basis of characteristic clinical features and investigated the exsome of these patients. METHODS: To evaluate disease genes, the Agilent SureSelect system and Illumina HiSeq 2000 platform for whole exome enrichment and sequencing (WES) were used on the proband and her mother. Variants that fit a recessive model of inheritance only were compared and filtered using public databases. Variants detected by exome sequencing were validated by Sanger sequencing. A total of 981 phenotypically normal subjects were enrolled as control data set. RESULTS: A frameshift homozygous germline mutation in BBS7 was detected by WES and identified by Sanger sequencing in affected individuals. This mutation was predicted to result in premature termination of exon5 (c.389_390delAC, p.Asn130ThrfsX3; RefSeq NM_176824.2) and lead to a 133 amino acid truncated protein. The inheritance patterns in the families are consistent with autosomal recessive inheritance, and no such homozygous mutation was found in the other 981 controls. CONCLUSION: This mutation has not yet been described in any reported literature, and this is the first report on BBS7 mutation in Chinese Miao families with BBS phenotypes.

Analysis of SLC4A11, ZEB1, LOXHD1, COL8A2 and TCF4 gene sequences in a multi-generational family with late-onset Fuchs corneal dystrophy.

The aim of the present study was to determine the genetic basis of a multi-generational family with late-onset (LO) Fuchs corneal dystrophy (FCD). Five FCD causal genes [solute carrier family 4, sodium borate transporter, member 11 (SLC4A11), zinc finger E-box binding homeobox 1 (ZEB1), lipoxygenase homology domains 1 (LOXHD1), collagen, type VIII, alpha 2 (COL8A2) and transcription factor 4 (TCF4)], previously reported to be implicated in the pathogenesis of FCD, were screened. A total of 27 variants [including 22 known single nucleotide polymorphisms (SNPs) from the Single Nucleotide Polymorphism Database (dbSNP) and 5 variants absent from dbSNP] were detected in this FCD pedigree across the SLC4A11, ZEB1, LOXHD1 and COL8A2 genes as follows: i) 22 known SNPs from dbSNP, including 3 coding (p.R161R, p.S213S and p.T833T) and 11 non-coding variants of SLC4A11, 2 intronic SNPs of ZEB1 from dbSNP (rs220057 and rs220060), 1 intronic SNP of LOXHD1 from dbSNP (rs16939650), and 5 SNPs of COL8A2 from dbSNP (p.A35A, p.R155Q, p.L335L, p.G495G and p.T502M); and ii) 5 variants that have not been previously reported in FCD patients and that are absent from dbSNP were identified across the ZEB1 and LOXHD1 genes; these included 3 continuous indels located at the junction of the 5'-UTR and the adjacent exon 1 of ZEB1 [Indel 1 (c.-86_-53delins gggaggggtggaggcggaggggtGGGGGGGAAGG); Indel 2 (c.-52_-46delinsGGGAGGG); and Indel 3 (c.-45_-42delinsAGGG)], and 2 intronic variants of LOXHD1 (c.5332-126C>T and c.1809+155G>A). Apart from one intronic SNP of SLC4A11 from dbSNP (rs372201212), the pathologic consequence of which is uncertain, and 2 intron variants of LOXHD1 (c.5332-126C>T and c.1809+155G>A); the variants likely represent examples of de novo mutations. Neither of the other 24 variants provided strong evidence of pathogenesis in this FCD pedigree. An analysis of 7 SNPs in TCF4 from dbSNP, which have been associated with LO FCD in different populations, revealed that these 7 SNPs were not associated with FCD in this specific pedigree. A genome­wide linkage scan to search for linkage to one of the previously described FCD loci or to identify a novel locus for FCD will need to be performed in this FCD pedigree. Our observation, nevertheless, expands the knowledge of the genetic status of patients with FCD.

Hepatitis B virus genetic mutations and evolution in liver diseases.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length. Owing to a lack of proofreading capacity during reverse transcription and a high replication rate, HBV exhibits as quasispecies. To detect the genetic mutations of HBV, many methods with different sensitivities and throughputs were developed. According to documentary records, HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome. In this review, we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression. Since some of the results were controversial from different laboratories, it remains to be seen whether functional analyses will confirm their role in modifying the course of infection.

Evolutionary perspective on hepatitis B virus with an expanded sampling strategy.

To investigate the role hepatitis B e antigen (HBeAg) plays in the evolution of hepatitis B virus (HBV), we sequenced the basic core promoter (BCP) and precore (preC) regions of 348 clones total from ten HBV Chinese patients. Eleven mutations were more frequent in HBeAg-negative patients than in HBeAg-positive patients. Further, the sequencing of dozens of variants was found to be necessary to obtain mutation profiles. Phylogenetic and median-joining network analyses suggested that variants from each patient had a single common ancestor (monophyly). Higher haplotype and nucleotide diversities were identified in HBeAg-negative patients. Analysis of dN/dS suggested that viruses experiencing a stronger immune response had lower haplotype diversity. Because HBeAg seroconversion was associated with viral diversity it served as an indicator of HBV evolution. Significantly, this study indicated a larger sampling of variants from each patient was required to understand effectively the properties of HBV.

[Current status and implication of research on Bardet-Biedl syndrome].

OBJECTIVE: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disease initially reported by Bardet and Biedl in the 1920s. BBS is a pleiotropic and genetically heterogeneous disorder characterized by retinopathy, obesity, polydactyly, renal malformations and functional abnormalities, learning disabilities and hypogenitalism. BBS patients are also prone to diabetes mellitus, hypertension and congenital heart disease. To date, 16 BBS genes (BBS1-BBS16) have been identified. However, the molecular etiology of BBS is not yet entirely clear. In this article, we have reviewed recent research on BBS and discussed its implications for understanding of ciliopathology.

An outbreak of sheep pox associated with goat poxvirus in Gansu province of China.

Three strains of Capripoxviruses (CaPVs) were isolated from an outbreak of sheep pox in Gansu province of China. They were analyzed by P32 gene-based molecular methods and a species-specific PCR based on the RPO30 gene. Two bands which are specific to goat poxvirus (GTPV) were observed after the PCR products of P32 gene were digested with the endonuclease of Hinf I. Moreover, an amplicon of 172 bp, which is specific to GTPV, was amplified from the viruses by using the RPO30 gene-based PCR. Sequence analysis of the P32 genes showed that three nucleotide bases for coding residue of aspartic acid which are located at 163-165 position of P32 gene of sheep poxvirus (SPPV) were absent, and six single nucleotide substitutions which are characteristic of GTPV were present. The viruses were genetically closer to GTPV strains and clustered into the GTPV branch of the phylogenetic tree constructed on the basis of the P32 gene. The results characterized the isolated viruses as GTPV. It is the first report of an outbreak of sheep pox associated with GTPV in China.

Evolution of Hepatitis B Virus in a Chronic HBV-Infected Patient over 2 Years.

Mutations in full-length HBV isolates obtained from a chronic HBV-infected patient were evaluated at three time points: 1 day, 6 months, and 31 months. While 5 nucleotides variation, and an 18 bp deletion of preS1 have been kept in during at least the first two years, C339T mutation occurring in the hydrophilic region of HBsAg and T770C that caused polymerase V560A substitution were the new point mutations found existing in sequenced clones of the 3rd time point. Internal deletion of coding region obviously appeared in the 3rd time point. The splicers included two new 5'-splice donors and three new 3'-splice acceptors besides the reported donors and acceptors and may have produced presumptive HBV-spliced proteins or truncated preS proteins. ALT, HBeAg and viral DNA load varied during the follow-up years. These data demonstrated the diversity of genomes in HBV-infected patient during evolution. Combined with clinical data, the HBV variants discovered in this patient may contribute to viral persistence of infection or liver pathogenesis.

[Establishment of B lymphoblastoid cell lines of Miao pedigree with Bardet-Biedl syndrome].

OBJECTIVE: To establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research. METHODS: With Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree, and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes. RESULTS: Twelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully. CONCLUSION: The immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.

[Effects of human bone marrow mesenchymal stem cells on cytokines secretion from allogeneic dendritic cell activated cytokine-induced killer cells].

AIM: to study the effect of human bone marrow derived mesenchymal stem cells (hMSCs) on cytokines secretion (IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2) of allogeneic DC-CIK cells (in co-culture of CIK cells with DC), which investigate the mechanism of immunoregulation induced by hMSCs. METHODS: the hMSCs from bone marrow were isolated, expanded and identified by cell morphology, differentiation into neuron-like cells with NSE, fat-like cells with red-oil stain, and expression of CD29, CD44. The DC and CIK cells from peripheral blood were isolated, expanded and identified by CD1α, HLA-DR or CD3(+);CD56(+);. The hMSCs were co-cultured with DC-CIK cells according to ratio 1:10. The expression of the six cytokines in supernatant was evaluated by flow cytometry after 4 days of DC-activated CIK cells in co-culture with hMSCs. RESULTS: the hMSCs displayed a fibroblast-like morphology and the positive cells of CD29 and CD44 were 96.6%, 94.6%, which have the capacity of differentiation into neuron-like cells with expressed NSE as well as fat-like cells with red-oil stain positive. The expression of CD1α, HLA-DR in DC was (91.9 ± 10.04)% and (88.8 ± 8.92)%. The CD3(+);CD56(+); double positive cells in DC-CIK cells was (29.23 ± 12.23)% compared to CIK cells with (15.98 ± 2.49)%. The cytokines secretion of DC-CIK cells in co-culture with hMSCs was IFN-γ (135.05 ± 48.19) ng/L; TNF-α (11.33 ± 1.42) ng/L; IL-10 (10.15 ± 2.25) ng/L; IL-6 (494.63 ± 235.222) ng/L; IL-4 (7.07 ± 2.30) ng/L and IL-2 (1074.6 3 ± 303.74) ng/L. In control group (DC-CIK cells) the secretion of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 was (717.6 ± 248.15) ng/L; (17.78 ± 7.52) ng/L; (29.95 ± 12.76) ng/L; (8.03 ± 0.21) ng/L, (9.08 ± 3.07) ng/L as well as IL-2 1 250 ng/L. CONCLUSION: the secretion of IFN-γ and IL-10 were down-regulated. It probably implied that hMSCs had the effect of immunoregulation on DC-CIK cells.

Identification of an intermediate signature that marks the initial phases of the colorectal adenoma-carcinoma transition.

The colorectal adenoma-carcinoma sequence describes the stepwise progression from normal to dysplastic epithelium and then to carcinoma. Only a small proportion of colorectal adenomas (CRAs) progress to colorectal carcinomas (CRCs). Endoscopic intervention is currently being used on patients with high grade dysplasia CRAs, with diameters of >1 cm, or villous components of >25% who are at higher risk than other CRA sufferers. During the process, biopsy samples are taken for conventional histological diagnosis, but poor pathomorphological sensitivity and specificity greatly limit the diagnostic accuracy. Unfortunately, there are no reliable molecular criteria available that can predict the potential development of CRA to CRC. Gene expression profiles of normal colorectal mucosa (NOR), CRA and different Dukes' stages of CRC biopsy specimens, which represent the gradual progress of the CRA to CRC sequence, were determined by Affymetrix technology. Representative regulated genes were further analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Intersectional analyses of discriminative expression signatures of CRC vs. CRA and CRA vs. NOR allowed the identification of an intermediate signature of 463 probe sets (psets) that mark the NOR--> CRA-->CRC progression. This signature represents a reservoir of candidate markers for the early diagnosis of higher-risk CRA, thus allowing for timely therapeutic intervention and more selective treatment. A further 279 CRC-specific psets pointing to the malignant transition from CRA to CRC were identified and these could represent potential therapeutic targets for CRC. The reliability of the results was further confirmed by qRT-PCR and IHC analyses of the 4-gene sets randomly selected from the 463 psets.

[Study on the relationship between HBV infection and polymorphism of CIITA G-944C gene among Jinuo, Dai and Aini minority populations in Yunnan province].

OBJECTIVE: To explore the relationship between HBV infection and the genotypes and allele frequencies of CIITA G-944C gene polymorphism in three minority populations (Jinuo, Dai and Aini population) in Xishuangbanna district, Yunnan province. METHODS: Polymerase chain reaction and sequencing method were used to study the genotypes and allele frequencies distributions of CIITA G-944C gene polymorphism in those three populations. Relationship between the genotypes distribution and HBV infection results were also analyzed. RESULTS: The rates on HBV infection and HBsAg carrier status in Aini minority population were 89.2% and 16.3%, which were significantly higher than in Jinuo (27.9% and 3.9%, χ(2) = 135.196 and 10.361, P = 0.000 and 0.001) and Dai population (44.9% and 6.6%, χ(2) = 96.783 and 8.748, P = 0.000 and 0.003) while among Aini population it was significantly different with the other two minority populations. The CC genotype and C allele frequencies were more distributed in Aini population than in the other two minority populations. In contrast, the GG genotype and G allele frequencies were lower than the other two minority populations, with χ(2) rates between Aini and Jinuo population were 11.841 and 12.208 and the P as 0.003 and 0.000 respectively while the χ(2) rates between Aini and Dai population were 23.902 and 20.220 with P value as 0.000 and 0.000. The genotypes frequencies of CIITA G-944C was significantly different in the infected individuals (IF) group and health control (HC) group in Jinuo population (χ(2) = 6.150 and 4.911, P = 0.046 and 0.027). When compared with HBsAg(+) group and HBsAg(-) group, the genotypes and allele frequencies were different in Aini population and the total three minority populations (χ(2) rates in Jinuo minority were 8.650 and 5.034 with P values as 0.013 and 0.025). However, the χ(2) rates in the whole population were 13.047 and 9.416 with P values as 0.001 and 0.002, respectively. The distribution of CC genotype and C allele gene in HBsAg(+) group was increasing. Data from non-condition logistic regression analysis and adjusting for confounding factors, the HBsAg(+) group had a significantly increase of HBsAg(-) group under the C allele Recessive Model (P = 0.000; OR = 2.964; 95%CI: 1.609 - 5.460). CONCLUSION: The genotypes and allele frequencies distribution of CIITA G-944C were different in the three ethnic populations. Polymorphism of this gene was closely associated with HBsAg carrier. The CC genotype patients were more easily to become HBsAg carrier.

Novel hepatitis B virus subgenotype in the southern Yunnan Province of China.

Hepatitis B virus (HBV) infection is highly prevalent in China. To identify the genotypes of HBV in the southern Yunnan Province of China, full-length HBV genomes were extracted from 1 Dai and 4 Hani HBV carriers and linked with the pMD T-18 vector. For each patient, 3-10 clones were sequenced directly and a consensus sequence was created. Genotypic and serotypic analysis revealed 4 HBV/B (2 B2 with adw2 and 2 new subgenotypes with ayw1) and 1 HBV/C (C1 with adrq+) genotypes. The divergences of the entire genome sequences of the new subgenotype were 0-0.9% and 2.99-6.48% between other known HBV/B. Divergences in other coding regions revealed that it was more similar to B3 and B4 in the precore/core gene (2.02 and 2.09%, respectively), and similar to B3 and B5 in the preS1/S2/S gene (2.24 and 2.78%, respectively). Phylogenetic trees using the precore/core and X genes both revealed a new clad separating from the major trunk of genotype B with a 99% bootstrap value. These results show that the 2 consensus isolates are a mosaic of B3-B5, which we designated to subgenotype B6. Considering the geographical distances, the relationship between B6 and other HBV/B subgenotypes (B3-B5) and HBV evolution needs to be further studied.

[Effect of liver X receptors agonists T0901317 on expression of FAT/CD36 gene mRNA in adult human skeletal muscle cell].

AIM: To investigate the liver X receptors agonists T0901317's effect on expression of FAT/CD36 gene mRNA in adult human skeletal muscle cell. METHODS: Myotubes from humans were exposed to different T0901317 concentrations (0, 0.5, and 1.0 micromol/L) for 24 hours before experiments were performed. Then the expression of FAT/CD36 mRNA in skeletal muscle cell of each experimental group was detected by SYBR Green I real-time quantitative polymerase chain reaction. The relative data were compared among groups by 2-delta delta Ct method. RESULTS: (1) The Ct mean of control group, T0901317 (0.5 micromol/L) group, T0901317 (1 micromol/L) group were analyzed and there was significant difference (P < 0.01). (2) The expression of FAT/CD36 mRNA with liver X receptors agonists T0901317 in human skeletal muscle cell in the T0901317 (0.5 micromol/L) group and T0901317 (1 micromol/L) group were 2.91 times and 3.03 times than the control group. CONCLUSION: The expression of FAT/CD36 mRNA in human skeletal muscle cell afer the treatment of liver X receptors agonists T0901317 is increased, so we may propose that T0901317 may increase the risk of resistance in adult human skeletal muscle.

Virologic characteristics of hepatitis B virus in patients infected via maternal-fetal transmission.

AIM: To determine whether HBV with the same characteristics causes dissimilar mutations in different hosts. METHODS: Full-length HBV genome was amplified and linked with pMD T18 vector. Positive clones were selected by double-restriction endonuclease digestion (EcoRI and HindIII) and PCR. Twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (S)], and the phylogenetic and mutational analysis was performed using BioEditor, Clustal X and MEGA software. Potential immune epitopes were determined by the Stabilized Matrix Method (SMM), SMM-Align Method and Emini Surface Accessibility Prediction. RESULTS: All of the 27 sequences were genotype C, the divergence between the mother and son was 0%-0.8%. Compared with another 50 complete sequences of genotype C, the mother and her son each had 13 specific nucleotides that differed from the other genotype C isolates. AA 1-11 deletion in preS1 was the dominant mutation in the mother (14/18). The 1762T/1764A double mutation existed in all clones of the mother, 3 of them were also coupled with G1896A mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential HLA class I epitopes and one B cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the P gene. CONCLUSION: The HBV strain in the son came from his mother, and discrepant mutation occurred in the mother and her son during infection.