Investigation of the molecular genetic causes of non-syndromic primary ovarian ınsufficiency by next generation sequencing analysis

ABSTRACT Objective: The aim of this study is to investigate the molecular genetic causes of non-syndromic primary ovarian insufficiency (POI) cases with the gene panel based on next generation sequencing analysis and to establish the relationship between genotype and phenotype. Subjects and methods: Twenty three cases aged 14-40 years followed up with POI were included. Patients with a karyotype of 46, XX, primary or secondary amenorrhea before the age of 40, with elevated FSH (>40 IU/mL) and low AMH levels (<0.03 ng/mL) were included in the study. Molecular genetic analyzes were performed by the next generation sequencing analysis method targeted with the TruSightTM Exome panel. Results: Median age of the cases was 17.8 (14.0-24.3) years, and 12 (52%) cases admitted before the age of 18. Fifteen (65%) patients had consanguineous parents. In 2 (8.6%) cases, variants detected were in genes that have been previously proven to cause POI. One was homozygous variant in FIGLA gene and the other was homozygous variant in PSMC3IP gene. Heterozygous variants were detected in PROK2, WDR11 and CHD7 associated with hypogonadotropic hypogonadism, but these variants are insufficient to contribute to the POI phenotype. Conclusion: Genetic panels based on next generation sequencing analysis technologies can be used to determine the molecular genetic diagnosis of POI, which has a highly heterogeneous genetic basis.

Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent / 中国输血杂志

【Objective】 To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i. e. Ion Torrent S5 and Illumina Miseq. 【Methods】 A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company; 336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. 【Results】 Ambiguous results were found in 357 out of 434 samples, accounting for 82.3% (357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8% (275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7% (82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing; HLA-DPB1*13∶01∶01 were detected in 25 (58.1%, 25/43) and HLA-DPB1*107∶01 in 18 (41.9%, 18/43). 【Conclusion】 There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1*13∶01∶01/107∶01 alleles.

Assessing multiple factors affecting the gut microbiome structure of very preterm infants

The composition and diversity of the gut microbiota are essential for the health and development of the immune system of infants. However, there is limited information on factors that influence the gut microbiota of very preterm infants. In this study, we analyzed factors that affect the gut microbiota of very preterm infants. The stool samples from 64 very preterm infants with a gestational age less than 32 weeks were collected for 16S rRNA gene sequencing. The infants were divided according to the delivery mode, antibiotic use during pregnancy, and feeding methods. The abundance of Proteobacteria was high in both cesarean (92.7%) and spontaneous (55.5%) delivery groups and then shifted to Firmicutes after the first week of birth. In addition, Proteobacteria was also the dominant phylum of infant gut microbiome for mothers with antibiotic use, with more than 50% after the first week of birth. In comparison, the dominant phylum for mothers without antibiotic use was Firmicutes. Proteobacteria level was also high in breastfeeding and mixed-feeding groups, consisting of more than 90% of the community. By contrast, Proteobacteria was the dominant phylum at the first week of birth but then shifted to Firmicutes for the formula-fed group. The alterations of gut microbiota in infants can affect their health condition during growth. This study confirmed that the different feeding types, delivery modes, and use of antibiotics during pregnancy can significantly affect the composition of the gut microbiota of very preterm infants.

Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae / Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae

Resumen Los hongos dematiáceos son un grupo heterogéneo de microorganismos capaces de sintetizar melanina. Las infecciones de este grupo que producen hifas en tejidos se denominan feohifomicosis y generalmente afectan la piel y tejidos vecinos. Presentamos el caso de un varón de 86 años con un tumor quístico blando progresivo en su mano y muñeca derecha, no asociado a dolor o signos inflamatorios. Se demostró una tenosinovitis de los flexores con pseudocapsula y sinovitis adherida a los tendones. El cultivo demostró un hongo dematiáceo compatible con Pleurostomophora richardsiae que se confirmó por secuenciación de la región ITS. La biopsia mostró una inflamación crónica granulomatosa e hifas. Después del drenaje quirúrgico, el paciente fue dado de alta sin terapia antifúngica, pero falleció por causas no relacionadas, tres meses después. Esta es la primera descripción de P. richardsiae como causa de feohifomicosis en Chile. Esta patología se puede sospechar cuando una lesión quística cutánea crónica involucra extremidades sin signos inflamatorios. Puede afectar a pacientes inmunocompetentes o inmunocomprometidos. El tratamiento contempla la escisión quirúrgica con o sin terapia antifúngica.

Abstract Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Characteristics of cervical microecology in late reproductive-age women with different grades of cervical lesions / 南方医科大学学报

OBJECTIVE@#To analyze the characteristics of cervical microecology in late reproductive-age women with cervical lesions and explore new methods for preventing cervical lesions.@*METHODS@#Cervical smears were obtained from a total of 147 women of late reproductive age, including 24 with high-risk HPV infection (HR-HPV), 27 with low-grade squamous intra-epithelial lesions (LSIL), 36 with high-grade squamous intra-epithelial lesions (HSIL), 35 with cervical cancer (CC) and 25 healthy women. llumina MiSeq sequencing of V3-V4 region of the 16S rRNA gene amplicons was used to characterize the vaginal microbiota of the women. OTUs analysis of the valid data was performed, and the α-diversity (Chao1, Simpson's Index and Shannon Index) and β-diversity (T-test, weighted UniFrac β diversity, and MetaStat analysis) were evaluated.@*RESULTS@#Dilution curve and species accumulation boxplot validated the quality of the samples. OTUs analysis of the 5 groups demonstrated that cervical bacterial genus consisted primarily of @*CONCLUSIONS@#The abundance of

Reprogramming of Cancer Cells into Induced Pluripotent Stem Cells Questioned

BACKGROUND AND OBJECTIVES: Several recent studies have claimed that cancer cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, in most cases, cancer cells seem to be resistant to cellular reprogramming. Furthermore, the underlying mechanisms of limited reprogramming in cancer cells are largely unknown. Here, we identified the candidate barrier genes and their target genes at the early stage of reprogramming for investigating cancer reprogramming.METHODS: We tried induction of pluripotency in normal human fibroblasts (BJ) and both human benign (MCF10A) and malignant (MCF7) breast cancer cell lines using a classical retroviral reprogramming method. We conducted RNA-sequencing analysis to compare the transcriptome of the three cell lines at early stage of reprogramming.RESULTS: We could generate iPSCs from BJ, whereas we were unable to obtain iPSCs from cancer cell lines. To address the underlying mechanism of limited reprogramming in cancer cells, we identified 29 the candidate barrier genes based on RNA-sequencing data. In addition, we found 40 their target genes using Cytoscape software.CONCLUSIONS: Our data suggest that these genes might one of the roadblock for cancer cell reprogramming. Furthermore, we provide new insights into application of iPSCs technology in cancer cell field for therapeutic purposes.

Analysis for correlation between drug resistance and positive pattern of virulence genes in Acinetobacter baumannii / 临床检验杂志

Objective@#To investigate the drug resistance and positive virulence genes of Acinetobacter baumannii (A.baumannii) and analyze the correlation between drug resistance and the positive pattern of virulence genes. @*Methods@#A total of 67 strains of A.baumannii were collected and identified by matrix assisted laser desorption ionization time of flight mass spectrometry technology (MALDI-TOF MS). Drug susceptibility tests were carried out by turbidimetric and redox indicator method. The homology of A.baumannii stains was explored by cluster analysis. The 8 virulence genes including bacterial outer membrane protein (ompA), biofilm formation (adeH, csuA, pgaA), iron uptake system (basJ), phospholipase D (plcD), capsular positive phenotype (ptk) and regulation of quorum sensing system (abaI) were amplified by PCR and sequenced. The correlation between virulence genes and drug resistance in the 67 strains of A. baumannii was investigated. @*Results@#The positive rates of virulence genes ompA, adeH, csuA, pgaA, abaI, basJ, ptk and plcD were 94%, 100%, 94%, 99%, 93%, 96%, 82% and 99%, respectively. Among the 67 strains of A. baumannii, 3 genes were simultaneously detectable in 1 strain (1.5%), 5 genes were positive in 2 strains (3.0%), 6 genes were positive in 2 strains (3.0%), 7 genes were positive in 14 strains (20.9%) and all the 8 genes were positive in 48 strains (71.6%). Among the 48 strains with 8 positive virulence genes, the drug resistance rate of polymyxin was only 2.1%, but tetracycline was 58.2%, piperacillin and other 13 antibiotics was more than 80%. The 14 strains with 7 positive virulence genes showed more than 78% of resistance rate for all the tested antibiotics except for tetracycline and polymyxin. Cluster analysis showed that the 67 strains of A. baumannii were divided into 2 genotypes: A (41 strains) and B (26 strains). The 41 strains of A type were divided into A1 (27 strains) and A2 (14 strains) subtypes. The strains of A1 subtype were mainly from neurosurgery department (7 strains), ICU (5 strains) and pneumology department (3 strains). The strains of A2 subtype were mainly from pneumology department (3 strains), cardiothoracic surgery department (3 strains), ICU (2 strains) and neurosurgery department (2 strains). The 26 strains of B type were divided into B1 (19 strains) and B2 (7 strains). The strains of B1 type were mainly from ICU (7 strains), neurosurgery department (4 strains) and respiratory department (3 strains). The strains of B2 type were mainly from ICU (2 strains) and respiratory department (3 strains). @*Conclusion@#The cross infection from A. baumannii may present in our hospital. There was no correlation between drug resistance and positive pattern of virulence gene in Acinetobacter baumannii.

The effects of green tea (Camellia sinensis) flower extracton melanin synthesis in B16-F10 melanoma cells / 대한수의학회지

The present study observed the effects of a green tea (Camellia sinensis) flower extract (GTFE) on melanin synthesis in B16-F10 melanoma cells. GTFE exhibited antioxidant activity on 2,2-diphenyl-1-picrylhydrazyl and inhibited mushroom tyrosinase activity in a dose-dependent manner. Furthermore, GTFE significantly diminished α-melanocyte stimulating hormone (α-MSH) stimulated cellular melanin content and tyrosinase activity throughout the concentration range evaluated. Based on RNA sequencing analysis, differential gene expression patterns observed in α-MSH stimulated B16-F10 melanoma cells were normalized by the addition of GTFE. In particular, the expression levels of melanoregulin and tyrosinase genes which are key regulating genes in melanin synthesis were up-regulated by 3.5 and 3 fold respectively by α-MSH, and were normalized to control levels by the addition of GTFE. The results suggest that GTFE inhibits melanin synthesis in α-MSH stimulated B16-F10 melanoma cells by normalizing expression of genes that are essential for melanin synthesis. Overall, the results suggest that GTFE could be applied in the development of a whitening agent for the treatment of dermal hyperpigmentation.

Fasciola hepatica: Infection Status of Freshwater Snails Collected from Gangwon-do (Province), Korea

Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments.

Capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi / 中华微生物学和免疫学杂志

Objective To evaluate the capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi.Methods A total of 267 filamentous fungi isolates collected from clinical specimens were analyzed by ITS region sequencing analysis.Alignment of acquired sequences with known sequences in GenBank and MycoBank was conducted to identify the species of those isolates.Results ITS sequences of the 267 isolates were amplified successfully.Among these isolates, 53.9％ (144/267) were identified to species level and 44.2％ (118/267) to genus level.Only five isolates were failed to be identified at genus level as they shared >95％ homology in ITS sequence with multiple genera.Conclusion ITS region sequencing analysis is preferred for identification of clinical isolates of filamentous fungi at genus level for its high universality and great capability.When species-level identification is required, some informative DNA markers besides ITS region should be included accordingly.

Genomic sequencing analysis of Cryptococcus neoformans var grubii strains of two genotypes with dif-ferent virulence and selection of virulence-associated genes / 中华微生物学和免疫学杂志

Objective To analyze the genomic sequences of Cryptococcus neoformans var grubii strains of two genotypes with different virulence and to screen out the virulence-associated genes. Methods A clinical strain (IFM56800) with the strongest virulence and an environmental strain (IFM56731) with the weakest virulence were screened out for whole genome sequencing analysis. The results of sequencing analy-sis were comprehensively analyzed by using the method of comparative genomics. Genetic variations were ex-tensively screened by using the strategies of non-synonymous single nucleotide polymorphisms ( nsSNPs), nonsense SNPs and the insertions or deletions ( InDels) causing frameshift mutations. The filtered genes were sequenced in 20 experimental strains. The whole RNAs were extracted and then the full-length cDNAs were sequenced by using the rapid amplification of 5′ and 3′ cDNA ends (RACE) method. Results By whole genome sequencing, valid data with high coverage (127 times and 111 times) was obtained in both the environmental strain IFM56731 and the clinical strain IFM56800. The data of InDels and SNPs were statisti-cally analyzed, respectively. Six genes were chosen for further analysis based on the strategies of nonsense SNPs and the InDels causing frameshift mutations. The six genes were amplified and sequenced in all of the experimental strains, three of which were further analyzed with cDNA sequencing. Ultimately, the location and structure of CNAG_01032 gene were determined. The predicted nonsense mutation locus was verified to present in the actual mRNA. Conclusion The strategies of nonsense SNPs and the InDels causing frame-shift mutations showed high-efficiency in screening potential virulence-associated genes. The CNAG_01032 gene was screened out as a novel virulence-associated gene.

Study on identification of Cronobacter spp.species in 11 areas in China / 中华流行病学杂志

Objective To compare different methods on the identification of Cronobacter (C.) spp.species and to choose an optimum one.Methods Biochemical test,16S rRNA and fusA sequencing methods were carried out.Results When using the biochemical test,105 strains showed six different conditions but C.turicensis and C.universalis could not be effectively identified.Under the 16S rRNA sequencing analysis,all the strains were divided into 5 groups but C.sakazakii and C.malonaticus were tangled.Finally,all the strains were identified into 58 C.sakazakii,30 C.malonaticus,11 C.dublinensis,5 C.turicensis,1 C.muytjensii,under the fusA sequencing analysis.Conclusion Currently,fusA sequencing analysis seemed an effective method for identifying the species of Cronobacter.Since fusA sequencing analysis method was less intuitive,another method for rapid testing should be developed.

Study on identification of Cronobacter spp.species in 11 areas in China / 中华流行病学杂志

Objective To compare different methods on the identification of Cronobacter (C.) spp.species and to choose an optimum one.Methods Biochemical test,16S rRNA and fusA sequencing methods were carried out.Results When using the biochemical test,105 strains showed six different conditions but C.turicensis and C.universalis could not be effectively identified.Under the 16S rRNA sequencing analysis,all the strains were divided into 5 groups but C.sakazakii and C.malonaticus were tangled.Finally,all the strains were identified into 58 C.sakazakii,30 C.malonaticus,11 C.dublinensis,5 C.turicensis,1 C.muytjensii,under the fusA sequencing analysis.Conclusion Currently,fusA sequencing analysis seemed an effective method for identifying the species of Cronobacter.Since fusA sequencing analysis method was less intuitive,another method for rapid testing should be developed.

Monitoring of Fasciola Species Contamination in Water Dropwort by cox1 Mitochondrial and ITS-2 rDNA Sequencing Analysis

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.

Cloning and analysis of tissue expression of PGC-1αgene in Guangxi Bama mini-pigs / 中国实验动物学报

Objective To clone the coding sequence of Guangxi Bama mini-pig PGC-1αgene, and to analyze the expression of PGC-1αgene in various tissues of mini-pigs using RT-PCR and QRT-PCR techniques.Methods The PGC-1αgene coding sequence ( CDS) was amplified by PCR from the cDNA of longissimus muscle of Guangxi Bama mini-pig. The PCR products were inserted into pEASY-T5 vector, transfected E.coli, identified and sequenced.The PGC-1αgene expression in different tissues of the Bama mini-pigs was detected by RT-PCR and QRT-PCR assays.Results The PGC-1αgene CDS of Guangxi Bama mini-pig was cloned.It was 2391 bp in length.It had 99.9%homology with the reference sequence, and had two synonymous mutations that were C-A1105 and G-A1524.The expression level of PGC-1αgene was higher in the heart and kidney, followed by liver, subcutaneous fat and longissimus muscle, but the expression was not de-tected in pancreas of Guangxi Bama mini-pig.Conclusions We have successfully cloned the PGC-1αgene of Guangxi Bama mini-pig, and detected this gene expression in six tissues.The results of this study will provide a basis for studying the effect of PGC-1αon type 2 diabetes mellitus (T2DM) in Bama mini-pigs.

A Comparative Study of the Diagnostic Performance of the GENEDIA Avellino Corneal Dystrophy Mutation Detection Kit and Screening Master Mix and a Direct Sequencing Method to Detect Mutations in the TGFB1 Gene / 임상검사와정도관리

BACKGROUND: Mutations in the gene encoding transforming growth factor-beta induced (TGFBI) are associated with corneal dystrophies. We evaluated the diagnostic performance of the GENEDIA Avellino corneal dystrophy (ACD) mutation detection kit and GENEDIA corneal dystrophy screening master mix (Green Cross Medical Science Co., Korea) by comparing it with an in-house sequencing method. METHODS: The study group consisted of 40 patients with Avellino corneal dystrophy (ACD) and 40 patients suspected to suffer from ACD; 40 healthy individuals were used as the control. All samples used for this study were previously obtained. All results obtained using the kit were evaluated for sensitivity, specificity, and detection limit. RESULTS: The sensitivity of the GENEDIA ACD kit was 100.0% with a positive mean+/-2SD Ct (cycle threshold) value of 25.87+/-1.24 and an excellent coefficient of variation value of 0.02 in ACD group. All normal control samples were negative, indicating a specificity of 100% for the GENEDIA kit. The detection limit was set at a DNA concentration of >0.2 ng/microL. Direct sequencing results obtained using the GENEDIA master mix and the in-house method agreed for all 20 ACD samples. Additional R555W mutation detected in four ACD-suspected samples were suggestive of the diagnosis of granular corneal dystrophy type I. CONCLUSIONS: The GENEDIA ACD detection kit and master mix showed acceptable results, demonstrating high sensitivity and specificity, and may be considered for clinical application. Furthermore, the GENEDIA master mix was useful for the detection of mutations in exons 4 and 12 of the TGFBI gene.

Comparison of Direct Sequencing, PNA Clamping-Real Time Polymerase Chain Reaction, and Pyrosequencing Methods for the Detection of EGFR Mutations in Non-small Cell Lung Carcinoma and the Correlation with Clinical Responses to EGFR Tyrosine Kinase Inhibito

BACKGROUND: The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs). METHODS: Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21). RESULTS: Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods. CONCLUSIONS: All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.

Diamond-Blackfan Anemia Confirmed by RPS19 Gene Mutation Analysis: A Case Study and Literature Review of Korean Patients / 대한진단검사의학회지

Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplastic anemia that usually presents early in infancy and is inherited in up to 45% of cases. It is characterized by red cell aplasia, congenital anomalies, and a predisposition to cancer. Corticosteroids and red blood cell transfusions are the mainstays of therapy. We describe a case of 3-month-old infant who presented with severe anemia, elevated levels of HbF and adenosine deaminase and bilateral hydronephrosis, who was later confirmed as DBA by mutation analysis using the direct sequencing method. Direct sequencing analysis of RPS19 gene was performed with both cDNA and genomic DNA extracted from peripheral blood and a c.3G>A point mutation of exon 2 resulting in p.Met1Ile was identified in this patient. The patient showed an inadequate response to steroid therapy and a partial response to RBC transfusion with a follow-up Hb level of 8.3 g/dL on her last visit to the outpatient clinic. DBA is a genetically and phenotypically heterogeneous disease, and we have reviewed the clinical characteristics of 25 Korean patients thus far reported in the literature. To our knowledge, this is the first case of DBA confirmed by mutation analysis in Korea, and mutation identification using molecular method is recommended for confirmation of this genetically and phenotypically heterogeneous disease.

Sequence analysis and genotyping of genital Chlamydia trachomatis among patients with suspected-Neisseria gonorrhoeae / 中华微生物学和免疫学杂志

Objective To understand the prevalence rate of genital Chlamydia trachomatis among a population with suspected-Neisseria gonorrhoeae infection,the distribution of Chlamydia trachomatis genotypes,assess changes in omp1 sequences among patients with Neisseria gonorrhoeae and Chlamydia trachomatis coinfections.Methods Four hundred and one swabs were collected.Chlamydia trachomatis and Neisseria gonorrhoeae were detected by Roche Amplicor System.DNA were extracted from those samples and were amplified by nested PCR.PCR products were sequencing and analyzed by software Mega4.0.Results The prevalence of genital Chlamydia trachomatis infection,Neisseria gonorrhoeae infection and coinfection with genital gonorrhoea and genital chlamydia were 82.3%,24.2% and 21.7% each.Eight genotypes were identified in 73 sequences,including E(27.4%),G/Ga(23.3%),D/Da(16.4%),F(13.7%),J (11.0%),H(5.5%),B and K(each 1.4%).Sequencing analysis showed that 3 cases(4.1%) had missense mutation,including genotype D/Da,E,G/Ga.Genotypes F,H,J and K were more variable,however,most of them were silent mutation.Conclusion The prevalence rate of genital Chlamydia trachomatis among a population with Neisseria gonorrhoeae infection was high.The most common genotypes were genotype E,G/Ga,D/Da and F; Sequencing analysis has provided a tool for the molecular epidemiology of genital Chlamydia trachomatis infections.

Isolation and identification of arboviruses from mosquito pools in some regions of Liaoning province,China / 中华流行病学杂志

Objective To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province.Methods Mosquitoes were collected from Shenyang,Yingkou,Panjin,Jinzhou and Dandong cities of Liaoning province in 2006.Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells.The new isolates were identified using serological and molecular biological methods.Results 5410 mosquitoes were collected from the five cities in total.Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BI-IK-21 cell.Three isolates (LN0684,LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus.Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates.The identity of nucleotide sequence was between 91.2% and 94.7%,compared with other Banna virus strains.The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine).The identity of nucleotide sequence was 99.2%,when comparing with the strain of South Korea and was 95% to 99% with the strains fi'om Russia,mainland of China and Taiwan region.Conehmion Eight virus isolates,including three Banna virus,one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province.Banna virus and Getah virus were reported for the first time in Liaoning province,while Getah virus showed the highest nucleotide homology with the South Korea strains.