Spectrum of ß-Thalassemia Mutations in Some Areas of Guangxi Zhuang Autonomous Region of Southern China: A Study on a Pediatric Population Aged 0-15 Years.

ß-Thalassemia (ß-thal), one of the most common form of single-gene inheritable blood diseases in the world, is highly prevalent in southern China, especially in the Guangxi Zhuang Autonomous Region. To update the ß-thal mutation spectrum in this region, we performed hematological and genetic analyses on 888 ß-thal major (ß-TM), ß-thal intermedia (ß-TI) and ß-thal carrier patients, aged 0-15 years old, from different parts of Guangxi Province. We identified 55 genotypes and 18 ß-thal mutations. The codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (43.97%), codon 17 (A>T) (HBB: c.52A>T) (25.43%), -28(A>G) (HBB: c.-78A>G) (8.18%), IVS-II-654 (C>T) (HBB: c.316-197C>T) (7.85%) and codon 26 (G>A) (HBB: c.79G>A) (5.02%) were the five most common, accounting for more than 90.0%. The results of our study are providing an up-to-date ß-thal mutation spectrum in the 0-15-year-old pediatric population, which will help genetic counseling and prevention of ß-TM in mainland China's most endemic region, Guangxi Zhuang Autonomous Region.

[Relationship between genotypes and clinical phenotypes in patients from 7 hemophilia A families].

OBJECTIVE: To study the mutation types of factor VIII (FVIII) gene in patients from 7 hemophilia A (HA) families and the relationship between FVIII gene mutations and clinical phenotypes. METHODS: A total of 8 patients from 7 HA families were recruited. The activated partial thromboplastin time (APTT) and factor VIII coagulant activity (VIII:C) in these patients were measured. Polymerase chain reaction (PCR) was performed to analyze FVIII gene intron 1 and 22 inversions. For patients without the FVIII intron inversions, direct sequencing was performed to determine their mutation types and other related members of their families were also tested by PCR and sequencing to analyze the corresponding mutation sites. RESULTS: The ranges of APTT and VIII:C of the 8 patients were 91.6-131 seconds and 0.8%-2%, respectively. FVIII gene intron 22 inversion was not detected, while intron 1 inversion was detected in one patient. There were 5 types of mutations in FVIII gene detected in the remaining 7 patients, including 6 patients with mutations in exon 14 and 1 patient with mutation in exon 23; all of the 5 types of mutations were single nucleotide mutations. Among the detected mutations in FVIII gene, p.His1202LeufsX16 (c.3666delA) detected in one patient was found to be a previously unreported mutation in FVIII gene. CONCLUSIONS: FVIII gene exon 14 is a hot-spot mutation region and p.His1202LeufsX16 is found to be a novel mutation in FVIII gene.

[Effects of silver ion of silver nanoparticles on gap junctional intercellular communication of human skin cells].

OBJECTIVE: To further investigate the effects of silver ion (Ag+) dissolved from silver nanoparticles (AgNps) on gap junctional intercellular communication (GJIC) between HaCaT cells. METHODS: In this study, 20 000×g of 1 g/L AgNps suspension were centrifuged at 4 °C for 2 h, then the supernatant was collected as Ag(+) stock solution and measured by inductively coupled plasma mass spectrometry (ICP-MS); GJIC was detected by the scrape loading/dye transfer assay; connexin 43 (Cx43) protein and mRNA level were estimated by Western-blot and RT-PCR, respectively. RESULTS: Different concentrations of Ag(+) (0.01, 0.1 and 1.0 mg/L) did not affect the GJIC significantly. No notable changes were observed in expression of Cx43 protein and mRNA. CONCLUSION: The characteristics of Ag(+) and the effects on gap junctional intercellular communication between HaCaT cells may be different from those of AgNps.

A remarkably diverse and well-organized virus community in a filter-feeding oyster.

BACKGROUND: Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation. RESULTS: Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels. CONCLUSIONS: In this study, we generated a first "knowledge landscape" of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. Video Abstract.

[Application of STR genetic marker system in the detection of hemophilia A carriers in Guangxi, China].

OBJECTIVE: To establish a fast and simple genetic diagnosis technique based on a reliable, short tandem repeat (STR) genetic marker system for the detection of hemophilia A carriers in Guangxi, China. METHODS: Fluorescent PCR and capillary electrophoresis were used for allele genotyping at three intragenic/extragenic STR loci (F8Int13, DXS1073, and DXS9901) of FVIII gene in the members of 10 hemophilia A families in Guangxi, so as to evaluate the diagnostic efficiency of the STR genetic marker system for detection of hemophilia A carriers. Then the STR genetic marker system was used to detect hemophilia A carriers among examinees. RESULTS: In the 10 hemophilia A families, 11 confirmed female carriers had the same allele fragment lengths at the three STR loci (F8Int13, DXS1073, and DXS9901) as the probands. Of the 8 females examined, 5 had allele fragments at the three STR loci (F8Int13, DXS1073, and DXS9901) which were identical to those of the probands, and thus they were diagnosed as hemophilia A carriers. CONCLUSIONS: Genetic analysis at the three STR loci (F8Int13, DXS1073, and DXS9901) can be used to detect hemophilia A carriers rapidly and provide reliable basis for prenatal diagnosis of hemophilia A.

[Genetic Analysis and Prenatal Diagnosis of a Family with Hereditary Spherocytosis Caused by a Novel Compound Heterozygous Mutation of SPTB Gene].

OBJECTIVE: To investigate the clinical and genetic characteristics of a family with hereditary spherocytosis (HS), to clarify the cause of the disease, and to provide the basis for genetic counseling and prenatal diagnosis. METHODS: The clinical data of proband and his parents were collected, and HS-related pathogenic genovariation of the proband was detected by high throughput sequencing. Suspected pathogenic mutation sites were verified by PCR-Sanger sequencing, and the fetus were conceived by a proband mother underwent prenatal diagnosis. RESULTS: Clinical manifestations of the proband showed moderate anemia, mild splenomegaly, and jaundice (an indirect increase of bilirubin). The gene detection showed that the proband showed compound heterozygous mutations of SPTB gene c. 6095T > C (p.Leu2032Pro) and c. 6224A > G (p.Glu2075Gly), which was inherited from the asymptomatic mother and father, respectively. Both mutations were detected rarely in the common population. Prenatal diagnosis revealed that the fetus inherited a mutant gene of the mother. CONCLUSION: The compound heterozygous mutations of SPTB genes c.6095T>C (p.Leu2032Pro) and c.6224A>G (p.Glu2075Gly) were the causes of the family disease, which provides a basis for family genetic counseling and prenatal diagnosis. This report is the first one found in the HGMD,1000G and EXAC database, which provides an addition to the mutation profile of the SPTB gene.

Real-time isothermal detection of Abalone herpes-like virus and red-spotted grouper nervous necrosis virus using recombinase polymerase amplification.

Abalone herpes-like virus (AbHV) and Red-spotted grouper nervous necrosis virus (RGNNV) are two serious viruses that infect animal populations in aquaculture. Both viruses cause diseases associated with high mortality rates, resulting in dramatic economic losses in the aquaculture industry. There are currently no effective treatments for either of these two viral diseases. Thus, early, rapid, and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Traditional methods of diagnosis, such as virus culture, enzyme-linked immunoassay, and polymerase chain reaction (PCR), are either time consuming or require sophisticated temperature control devices. In this study, one sets of specific primers and probes were designed for the real-time quantitative recombinase polymerase amplification (qRPA) detection of AbHV and RGNNV separately. The sensitivity and specificity of detection were evaluated by comparison with detection by conventional PCR and quantitative PCR. The optimal reaction temperature and time for virus detection is 37°C for 20min. The detection limit is 100 copies per reaction, making this approach faster and more sensitive than qPCR in this study. In a field application, the detection percentage of qRPA was higher than that of qPCR for both AbHV and NNV. Additionally, good correlation was found between qRPA and qPCR detection (R2>0.8). The methods presented here can be used as alternatives to qPCR for quick and quantitative detection of pathogens infecting aquaculture species.

Real-time quantitative isothermal detection of Ostreid herpesvirus-1 DNA in Scapharca subcrenata using recombinase polymerase amplification.

Ostreid herpesvirus-1 (OsHV-1) is a well-known pathogen associated with high mortality rates in hatchery-reared larvae and juveniles of different bivalve species worldwide. Early, rapid and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Recombinase polymerase amplification (RPA) is a novel isothermal amplification method, which can amplify detectable amount of DNA at 37 °C-39 °C within 20 min. In the present study, two sets of specific primers and probes were designed for the real-time quantitative RPA (qRPA) detection of OsHV-1 DNA. The sensitivity and specificity of detection were evaluated by comparison with quantitative polymerase chain reaction (qPCR). The detection limit for qRPA assays was shown to be 5 copies DNA/reaction for the primer set ORF95, which was lower than the 100 copies required for the qPCR test. The optimal reaction temperature and time were 37 °C for 20 min, making this approach faster than qPCR. This is the first study to apply qPCR and qRPA methods to detect OsHV-1 in Scapharca subcrenata. The percentage of viral load sample detected by the two methods was 22% and the correlation of the two virus quantitative results was 0.8. Therefore, qRPA assays is sensitive, fast, and high-temperature independent relative to qPCR and is suitable for critical clinical diagnostics use and rapid field analysis in resource-limited settings.

Comparison of methods for library construction and short read annotation of shellfish viral metagenomes.

The emergence and widespread use of high-throughput sequencing technologies have promoted metagenomic studies on environmental or animal samples. Library construction for metagenome sequencing and annotation of the produced sequence reads are important steps in such studies and influence the quality of metagenomic data. In this study, we collected some marine mollusk samples, such as Crassostrea hongkongensis, Chlamys farreri, and Ruditapes philippinarum, from coastal areas in South China. These samples were divided into two batches to compare two library construction methods for shellfish viral metagenome. Our analysis showed that reverse-transcribing RNA into cDNA and then amplifying it simultaneously with DNA by whole genome amplification (WGA) yielded a larger amount of DNA compared to using only WGA or WTA (whole transcriptome amplification). Moreover, higher quality libraries were obtained by agarose gel extraction rather than with AMPure bead size selection. However, the latter can also provide good results if combined with the adjustment of the filter parameters. This, together with its simplicity, makes it a viable alternative. Finally, we compared three annotation tools (BLAST, DIAMOND, and Taxonomer) and two reference databases (NCBI's NR and Uniprot's Uniref). Considering the limitations of computing resources and data transfer speed, we propose the use of DIAMOND with Uniref for annotating metagenomic short reads as its running speed can guarantee a good annotation rate. This study may serve as a useful reference for selecting methods for Shellfish viral metagenome library construction and read annotation.

[Molecular analysis of two pedigrees with inherited coagulation factor VII deficiency].

OBJECTIVE: To analyze the gene mutation in two pedigrees of inherited coagulation factor VII (FVII) deficiency, and investigate the relationship between the genotype and phenotype. METHOD: The coagulation function and coagulation factors activity of probands were detected for phenotype diagnosis, all exons and junctions of FVII gene from the family members' genomic DNA were amplified using polymerase chain reaction (PCR), and detected the gene mutation by direct sequencing. Mutations were confirmed by reverse sequencing. RESULT: The prothrombin time (PT) of proband 1 was 265.2 s, FVII:C was 22% and the PT of proband 2 was > 120 s, FVII:C was 1%. Homozygous 17844G→A mutation in No. 8 exon of FVII gene was identified in the proband 1 resulting in Gly343Ser, and heterozygosity for the same mutations were confirmed in his parents and a sister. The proband 2 was compound heterozygous, one mutation was the same as the proband 1 but was a heterozygosity that can also found in his mother and brother; the other heterozygosity mutation was located on No. 8 exon 18055G→A that resulted in Gln413Arg which was inherited from his father. CONCLUSION: No. 8 exon of FVII gene encodes catalytic domain. Mutation found in those domain could change the FVII catalytic domain spatial structure, affected FVII function and stability, and the sufferer of homozygote and compound heterozygous may have clinical bleeding tendency. Almost no clinical findings in simple heterozygotes, however, a few of heterozygotes could have a tendency of bleeding because of genetic polymorphism which would reduce the FVII:C.

[Non-invasive prenatal genetic diagnosis of beta-thalassaemia using single fetal nucleated erythrocyte from maternal blood].

OBJECTIVE: To investigate non-invasive prenatal genetic diagnosis of beta-thalassaemia using a single fetal nucleated erythrocyte (NRBC) from maternal blood by comparing with the genotype obtained from chorionic villus or amniocytes, and to evaluate the diagnostic results in reliability and feasibility of this method. METHODS: Maternal blood samples were obtained from 28 pregnant women at risk of beta thalassaemia during 9 - 34 weeks of gestation. NRBCs in maternal blood were enriched by single density gradient separation, stained with benzidine, and then collected by micromanipulation individually. After primer extension preamplification (PEP) of the entire genome from each single NRBC, short tandem repeat (STR) genotype was analyzed after further amplification of this gene. Single NRBC was tested individually to identify if it was fetal or maternal in origin by STR genotype of NRBC and its corresponding parents. beta-globin DNA fragments were amplified with nested-PCR using PEP product of a single fetal NRBC that was determined to be fetal in origin. Fetal beta-globin genotypes were analyzed by reverse dot-blot hybridization (RDB), the accuracy was evaluated by comparing with genotype which had been determined on DNA obtained from chorionic villus (CVS) or amniocytes. RESULTS: A total of 298 NRBCs were found in all of 28 pregnant women at a range of 4 to 13 per 5 ml venous blood. After PEP, about 43.6% of NRBCs were determined to be fetal in origin by STR typing. Using PEP product of a single fetal NRBC as template, beta-globin DNA fragment was examined on agarose gel after nested-PCR, amplification efficiency was 90.8% (118/130). Fetal beta-globin genotypes were achieved successfully in all cases with RDB. Comparing with the genotypes which were obtained from CVS or amniocytes, the rate of diagnostic accuracy was 85.7% (24/28). CONCLUSIONS: PEP technique and STR genotype analysis provide effective method for identification of single nucleated erythrocyte from maternal blood in origin. With the techniques PEP and RDB, fetal beta-globin genetic diagnosis was achieved using a single fetal NRBC from maternal blood. The method had a high accuracy and reliability in diagnosis. It may become an optional approach to non-invasive prenatal diagnosis of beta-thalassemia.

[Identification of fetal nucleated erythrocytes in maternal blood using short tandem repeat typing after primer extension preamplification].

OBJECTIVE: To develop a method for identifying fetal nucleated erythrocytes (NRBCs) in maternal blood. METHODS: NRBCs in maternal blood were detected by benzidine staining and collected by micromanipulation. After primer extension preamplification (PEP) of the entire genome from a single NRBC, short tandem repeat (STR) genotype was analysed after further amplification of this gene. Single NRBC was differentiated as fetal or maternal origin by comparison of STR genotype of NRBC with its corresponding parents. RESULTS: NRBCs were found in all of 28 pregnant women in a range of 4 to 13 per 5 ml venous blood. About 43. 6% of NRBCs were determined to be fetal origin by STR typing. CONCLUSION: This method provides effective identification of fetal NRBCs and allows non-invasive prenatal genetic diagnosis using single fetal NRBC.

[Relationship between the expression of murine double minute 2 oncogene and non-Hodgkin lymphoma in childhood].

OBJECTIVE: To investigate the relationship between the expression of murine double minute 2 (MDM2) oncogene and non-Hodgkin lymphoma (NHL) in childhood. METHODS: Thirty-one cases of NHL were enrolled in this study as patient group and 8 cases of lymphadenitis as control group. (1) Immunohistochemistry ultrasensitive S-P assay was used to detect the expression of MDM2 protein in pathological tissues in all cases. Positive cells were dyed yellow or brown in nuclei. MDM2 positive cell was defined as >/= 10% of the tumor cells were positive, which was overexpression of MDM2 protein. (2) RT-PCR (reverse transcription-polymerase chain reaction) was performed to value the overexpression of MDM2 mRNA in the pathological tissues and mononuclear cells in peripheral blood. While the ratio of MDM2/beta-actin was >16% was defined as overexpression of MDM2 mRNA. RESULTS: (1) Rates of overexpression of MDM2 protein and MDM2 mRNA were 64.5% and 61.3%, respectively, which were significantly different as compared to that of control group (P < 0.05 and P < 0.01, respectively). (2) The relationship analysis among subgroups in the experiment group showed that the overexpression of MDM2 protein did not correlate with classifications of working formulation, cellular origin, sex, clinical stage and involved extranodal sites (P > 0.05), but significantly correlated with classifications of B status and the increased serum LDH level (P < 0.05). It was shown that the overexpression of MDM2 mRNA did not correlate with classifications of working formulation, cellular origin, sex and clinical stage (P > 0.05), significantly correlated with B status (P < 0.05), and was remarkably significantly correlated with the involved extranodal sites and the increased serum LDH level (P < 0.01). (3) It was demonstrated that the overexpression of MDM2 mRNA in the pathological tissues was similar to the overexpression of MDM2 protein in the pathological tissues and MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.655 and 0.571), and the overexpression of MDM2 protein in the pathological tissues was similar to that of MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.609). CONCLUSIONS: (1) The rate of MDM2 oncogene overexpression was quite high. (2) The overexpression of MDM2 protein in pathological tissues determined by using immunohistochemistry ultrasensitive S-P assay was similar to that of MDM2 mRNA in pathological tissues detected by using RT-PCR method. Both methods might be used to detect the overexpression of MDM2 oncogene in the cases of childhood NHL. (3) The overexpression of MDM2 oncogene related to the poor status and poor prognosis of patients with childhood NHL.