Genomic DNA Samples Research Articles

Targeted next-generation sequencing for identifying genes related to horse temperament

It is a fundamental challenge to discover the association of genetic traits with phenotypic traits. In this study, we aimed to identify possible genetic traits related to horse temperament. Based on previous findings, we selected 71 candidate genes related to temperamental trait and examined them in the human and horse reference genomes (hg38 and equCab2, respectively). We found 16 orthologous genes and, by comparing with the human reference genome, 17 homologous genes in the horse reference genome. We designed probes specific for the 33 horse genes. Using the probes, we built sequencing libraries of the genomic DNA samples from eight aggressive and eight docile horses, and sequenced the constructed libraries using the Illumina Hiseq2500 platform. Through the analysis of the targeted exome sequences, we identified single nucleotide polymorphisms (SNPs) in the genes. SNPs could be served as genetic markers to evaluate aggressive or docile levels of horses. To examine whether any genetic variants are associated with horse temperament, we performed genome-wide association study (GWAS) using the SNP data. GWAS analysis identified ten variants (p-value C) and AR (c.1047+27G>T) genes with 8.09 × 10−4 p-value. We suggest that the variants might be used to assess horse temperament and to determine superior horses for riding or racing.

A novel and quick PCR-based method to genotype mice with a leptin receptor mutation (db/db mice)

db/db mice is one of most widely used animal models in studying the cellular and molecular mechanisms of metabolic disorders, such as diabetes, hyperlipidemia, and obesity. The mice carry spontaneous point mutations in the gene encoding the leptin receptor, leading to leptin receptor inactivation. Since homozygous db/db mice are sterile, the maintenance of db/db mice requires breeding between heterozygous pairs, which makes genotyping essential for the identification of offspring. The aim of this study was to develop a quick and highly repeatable method for genotyping db/db mice, which comprised only three simple steps: genomic DNA is extracted from either tail tips or ear notches via alkaline lysis (∼20 min); samples are then subjected to tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using specially designed and validated primer sets (∼1.5 h); finally, genotypes are be determined by resolving PCR products on regular DNA electrophoresis (∼10 min). The entire db/db mice genotyping procedure can be performed using regular Taq polymerase and PCR amplification within 2 h. Other advantages of this method include high sensitivity and reproducibility. Minimal amounts of tissue from mice are required, and genomic DNA samples can be stably stored at room temperature for up to one month. In conclusion, the method is simple, cost effective, sensitive and reliable, which will greatly facilitate studies using db/db mice.

Characterization of the Culturable Subpopulations of Lactobacillus in the Chicken Intestinal Tract as a Resource for Probiotic Development.

To gain better understanding of the distributions of the culturable Lactobacillus species in the chicken intestinal tract, we collected ceca, and distal ileum from 10 3-weeks-old broiler chickens. Lactobacillus strains from cecal lumen contents (M-CL), and those associated with mucosa of ceca (M-CM) and ileum (M-IM) were recovered on de Man, Rogosa and Sharpe (MRS) agar plates, and used for microbiota analysis. The total cecal content (T-CL) was also used directly for microbiota analysis. We purposefully focused on MRS-recovered populations to gain understanding of the culturable subpopulations of Lactobacillus, since the culturability is an important phenotype in order to exploit the chicken gut microbiota as a resource for development of probiotics. The V1–V3 regions of 16S rRNA gene was amplified from genomic DNA samples, and the pooled amplicons were analyzed by MiSeq sequencing with paired-end read 300 cycle option. Among MRS groups, Firmicutes were significantly higher in M-IM and M-CL as compared to M-CM, whereas Proteobacteria were significantly higher in M-CM as compared to M-IM and M-CL at p < 0.05. Among Lactobacillus, L. salivarius (36%) and L. johnsonii (21%) were higher in M-IM as compared to M-CL (L. salivarius, 28%; L. johnsonii, 15%), and M-CM (L. salivarius, 20%; L. johnsonii, 11%). L. crispatus was found significantly higher in M-CL as compared to M-IM (p < 0.01) whereas L. gasseri was found significantly higher in M-IM as compared to M-CM (p < 0.05). L. aviarius, and L. fornicalis were only observed in T-CL. In summary, Lactobacillus populations recovered on MRS vary with different regions and locations in chicken GIT, which might indicate their distinct functional roles in different gastrointestinal tract (GIT) niches, and some species of Lactobacillus are not culturable on MRS agar media. This study is the first attempt to define culturable Lactobacillus subpopulations in the chicken intestinal tract comprehensively using 16S rRNA gene profiling, and the findings of this study will be used as a platform to develop a new strategy for isolation of effective Lactobacillus probiotic candidates based on comparative analyses of chicken gut microbiota.

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling.

Somatic Mutations and Splicing Variants of Focal Adhesion Kinase in Non-Small Cell Lung Cancer.

Overexpression of focal adhesion kinase (FAK) has been reported in lung cancer, but the somatic mutations and alternative splicing variants of this nonreceptor tyrosine kinase remain to be investigated. FAK in 91 lung cancer patients was sequenced using genomic DNA and cDNA samples of tumor and paired normal lung tissues as templates, and the RNA-seq data of The Cancer Genome Atlas (TCGA) data set were assessed. The biological functions of abnormal FAK transcripts and their response to FAK inhibitors were analyzed in eight cell lines using tyrosine kinase activity assay, trypan blue exclusion assay, MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide) assay, and transwell assay. We identified an internal tandem duplication (ITD), an A1004S point mutation, an exon 5-27 deletion (ΔE5-27) truncation variant, and four FAK6,7 splicing variants (containing exons for Boxes 6 and 7) in seven (7.7%) patients. Smokers had more FAK abnormalities than nonsmokers. In FAK-ITD, the sequence encoding the C-terminal of the FERM domain and kinase domain was duplicated in-frame and produced a protein product with elevated autophosphorylation and sensitivity to FAK inhibitors. FAK6,7 was detected in the tumor but not counterpart normal lung tissues of four (4.4%) patients. In TCGA RNA-seq data, Box 6 and/or Box 7 (Box 6/7)-containing FAK variants were positive in 42 (8.3%) of 508 lung adenocarcinomas (LUADs) and 37 (7.4%) of 501 lung squamous cell carcinomas, and smokers had higher expression of Box 6/7 (+) FAK than reformed or nonsmokers with LUAD. FAK6,7 promoted cell proliferation and migration, exhibited increased autophosphorylation, and was more sensitive to FAK inhibitor compared with wild-type FAK. Somatic mutations and splicing variants of FAK may have a role in lung carcinogenesis and represent potential biomarkers for FAK-targeted therapies.

31 Effect of cntnap2 polymorphisms on cerebral response to human voice perception and handedness: an fmri study

<h3>Objective</h3> Contactin-associated protein-like2 (CNTNAP2) is one of the risk markers in the pathogenesis of language impairment and thought disturbance, such as autism spectrum disorders and schizophrenia. Although recent neuroimaging studies have demonstrated that CNTNAP2 polymorphisms affect left-hemispheric function of language processing, no study has investigated the influence of these polymorphisms on right-hemispheric function involved in human voice perception, and handedness. We investigated whether CNTNAP2 polymorphisms (rs7794745 and rs2710102) affect voice-specific brain function and handedness. <h3>Method</h3> One hundred and eight healthy Japanese volunteers (74 right-handed and 34 non-right-handed) had their brain function examined while they were passively listening to reverse sentences (rSEN), identifiable non-vocal sounds (SND), and sentences (SEN). The study protocol was approved by Ethics Committee. After complete explanation of the study, written informed consent was obtained from all subjects. Data analysis of fMRI was performed with statistical parametric mapping software SPM8. Genomic DNA samples were extracted from peripheral blood using standard procedures. Since these SNPs are known as biological high-risk markers for ASD, epilepsy, mental retardation, schizophrenia, and cognitive impairment, we investigated the genotype of brain function in these 2 SNPs: (rs7794745 and rs2710102) in CNTNAP2. The genotyping of all subjects was determined in comparison with control DNA confirmed by sequencing in the SNP pattern. Genotype of rs7794745 is A/A, A/T, and T/T; that of rs2710102 is G/G, G/A, A/A. The genotypes were classified into A/A and A/T in rs7794745, and G/G and A carriers (G/A and A/A) in rs2710102, respectively. <h3>Results</h3> In full factorial design analysis, rs7794745 (A/A or A/T) × rs2710102 (G/G or A carrier (A/G and A/A)) × voice-specific response (rSEN or SND), the main effect of rs7794745 (A/A or A/T) was significantly revealed at the right middle frontal gyrus (MTG) and bilateral superior temporal gyrus (STG). This result suggests that rs7794745 genotype impacts on voice- specific brain function. Furthermore, interaction effect was significantly observed among MTG-STG activations by human voice perception, rs7794745 (A/A or A/T), and handedness. <h3>Conclusion</h3> CNTNAP2 polymorphisms could be one of the remarkable biomarkers verifying genetic influence in voice-specific brain function and handedness in psychiatric disorders.

Evaluation of the relationship between MARCO and CD36 single-nucleotide polymorphisms and susceptibility to pulmonary tuberculosis in a Chinese Han population

BackgroundGene polymorphisms impact greatly on a person’s susceptibility to pulmonary tuberculosis (PTB). Macrophage receptor with collagenous structure (MARCO) and CD36 are two scavenger receptors (SRs) that can recognize Mycobacterium tuberculosis (Mtb) and play a key role in tuberculosis infection. Gene polymorphisms of MARCO and CD36 may contribute to tuberculosis risk.MethodsTo investigate whether genetic polymorphisms of MARCO and CD36 are associated with susceptibility to PTB, genomic DNA samples from patients (n = 202) and healthy controls (n = 216) were collected and analyzed by polymerase chain reaction with high-resolution melting analysis.ResultsWe studied two single nucleotide polymorphisms (SNPs) in MARCO (rs12998782 and rs17009726) and three SNPs in CD36 (rs1194182, rs3211956 and rs10499859). Rs12998782 (P = 0.018) might be associated with susceptibility to PTB. Rs1194182 (P < 0.01) and rs10499859 (P < 0.001) might be associated with resistance to PTB. Rs17009726 and rs3211956 were not associated with susceptibility/resistance to PTB.ConclusionsThese data showed that MARCO rs12998782 may increase PTB risk while two SNPs of CD36, rs1194182 and rs10499859 may reduce the risk, indicating MARCO and CD36 as important receptors in response to PTB.

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

Genetic variability of interleukin-1 beta as prospective factor from developing post-traumatic stress disorder.

Individual susceptibility to post-traumatic stress disorder (PTSD) is conditioned by genetic factors, and association between this disorder and polymorphisms of several genes have been shown. The aim of this study was to explore a potential association between single nucleotide polymorphisms (SNP) of the IL-1β gene (IL1B) and PTSD. In genomic DNA samples of PTSD-affected and healthy subjects, the rs16944, rs1143634, rs2853550, rs1143643, and rs1143633 SNPs of IL1B gene have been genotyped. The results obtained demonstrated that IL1B rs1143633*C and rs16944*A minor allele frequency were significantly lower in patients than in controls. Our results confirm that IL1B rs1143633 and rs16944 SNPs are negatively associated with PTSD which allows us to consider them as protective variants for PTSD. IL1B rs1143633*C and rs16944*A minor allele frequencies and carriage rates are significantly lower in the PTSD patients as compared to the controls. These results may provide a base to conclude that above-mentioned alleles can be protective against PTSD, and IL1B gene can be involved in the pathogenesis of this disorder.

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a promising tool for diagnosis of P. falciparum infection.

Abstract 1290: Association of common genetic variants with TMPRSS2 ERG fusion status in prostate cancer

Abstract Introduction and Objectives: Oncogenic activation of ERG resulting from prevalent gene fusions is present in two thirds of prostate cancer (CaP) patients of European Ancestry including Caucasian Americans (CA). Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status. The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. Methods: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center (WRNMMC). ERG status was determined in whole mounted prostate specimens by immuno-histochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. Results: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (&amp;lt;10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G protein-coupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. Conclusions: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options. Citation Format: Indu Kohaar, Lakshmi Ravindranath, Denise Young, Amina Ali, Qiyuan Li, Albert Dobi, David McLeod, Inger L. Rosner, Isabell Sesterhenn, Matthew Freedman, Shiv Srivastava, Gyorgy Petrovics. Association of common genetic variants with TMPRSS2 ERG fusion status in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2017-1290

Development of a Medium Density Combined-Species SNP Array for Pacific and European Oysters (Crassostrea gigas and Ostrea edulis).

SNP arrays are enabling tools for high-resolution studies of the genetic basis of complex traits in farmed and wild animals. Oysters are of critical importance in many regions from both an ecological and economic perspective, and oyster aquaculture forms a key component of global food security. The aim of our study was to design a combined-species, medium density SNP array for Pacific oyster (Crassostrea gigas) and European flat oyster (Ostrea edulis), and to test the performance of this array on farmed and wild populations from multiple locations, with a focus on European populations. SNP discovery was carried out by whole-genome sequencing (WGS) of pooled genomic DNA samples from eight C. gigas populations, and restriction site-associated DNA sequencing (RAD-Seq) of 11 geographically diverse O. edulis populations. Nearly 12 million candidate SNPs were discovered and filtered based on several criteria, including preference for SNPs segregating in multiple populations and SNPs with monomorphic flanking regions. An Affymetrix Axiom Custom Array was created and tested on a diverse set of samples (n = 219) showing ∼27 K high quality SNPs for C. gigas and ∼11 K high quality SNPs for O. edulis segregating in these populations. A high proportion of SNPs were segregating in each of the populations, and the array was used to detect population structure and levels of linkage disequilibrium (LD). Further testing of the array on three C. gigas nuclear families (n = 165) revealed that the array can be used to clearly distinguish between both families based on identity-by-state (IBS) clustering parental assignment software. This medium density, combined-species array will be publicly available through Affymetrix, and will be applied for genome-wide association and evolutionary genetic studies, and for genomic selection in oyster breeding programs.

Abstract 4254: Improved understanding of the etiology of breast cancer in young women through evaluation of the genomic landscape

Abstract Background Breast cancer is a disease where biological age and age at menopause are established risk factors, thus diagnosis of breast cancer in young women (&amp;lt;40 years, YW) is not common, representing ~6% of breast cancer diagnoses annually. Germline mutations in cancer predisposition genes accounts for ~20% of breast cancer diagnoses in YW, thus the etiology of breast tumors for the majority of YW is unknown. Methods Frozen tumor specimens and matched genomic DNA samples from 31 women diagnosed &amp;lt;40 years were available. Whole genome sequencing (WGS) was performed by generating sequencing libraries from 1 mg input using the TruSeq PCR-free library preparation workflow and sequencing on the HiSeq X Ten at triple lane depth with 150bp paired-end reads. WGS was analyzed using the Hiseq Analysis Software Tumor Normal Workflow, Bioconductor: deconstructSigs and ad hoc integrated analysis and hypothesis testing. Results Luminal A tumors from YW had significantly lower somatic mutational loads, copy number alterations and structural variants than other subtypes and structural variants on chromosome 17 were enriched in patients with the HER2-enriched subtype and HER2 status = 3+. Aging was the most common signature (28/31) followed by APOBEC activation (14/31). Signatures associated with defects in mismatch repair (9/31) and polymerase ϵ (10/31) were also detected in &amp;gt;25% of patient tumors. Of the six patients with BRCA defect signatures, three had germline mutations in BRCA2 and one had a CHEK2 mutation. Mutational signatures could cluster patients into two groups, with the youngest patients (&amp;lt;35 years) less likely to have signatures associated with aging but an enrichment in signatures of APOBEC, BRCA mutations and smoking; tumors within this group were more likely to be non-luminal A. Conclusions Profiling the genome of tumors from YW supports the idea that most tumors are not characterized by profiles associated with genetic predisposition but rather processes such aging, APOBEC activation, mismatch repair, and defects in DNA replication. Of note, breast tumors in the youngest patients (&amp;lt;35) have a different pattern of mutational signatures suggesting that even within YW, etiology differs by age. Citation Format: Matthew D. Wilkerson, Clifton L. Dalgard, Craig D. Shriver, Rachel E. Ellsworth. Improved understanding of the etiology of breast cancer in young women through evaluation of the genomic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4254. doi:10.1158/1538-7445.AM2017-4254

Genetic Analyses of the NF1 Gene in Turkish Neurofibromatosis Type I Patients and Definition of three Novel Variants.

Neurofibromatosis Type I (NF1) is a multi systemic autosomal dominant neurocutaneous disorder predisposing patients to have benign and/or malignant lesions predominantly of the skin, nervous system and bone. Loss of function mutations or deletions of the NF1 gene is responsible for NF1 disease. Involvement of various pathogenic variants, the size of the gene and presence of pseudogenes makes it difficult to analyze. We aimed to report the results of 2 years of multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) for genetic diagnosis of NF1 applied at our genetic diagnosis center. The MLPA, semiconductor sequencing and Sanger sequencing were performed in genomic DNA samples from 24 unrelated patients and their affected family members referred to our center suspected of having NF1. In total, three novel and 12 known pathogenic variants and a whole gene deletion were determined. We suggest that next generation sequencing is a practical tool for genetic analysis of NF1. Deletion/duplication analysis with MLPA may also be helpful for patients clinically diagnosed to carry NF1 but do not have a detectable mutation in NGS.

A multiplex restriction enzyme-PCR for unequivocal identification and differentiation of Trichostrongylus species in human samples

Trichostrongylus species remain one of the major health challenges in the tropical and summer rainfall regions worldwide. Identification of strongylid species diagnostic methods is vital for obtaining a deep understanding of the epidemiology, population biology, anthelmintic treatment efficacy, and drug resistance in order to design effective parasite control strategies. We evaluated a multiplex RE-PCR for the diagnosis of key Trichostrongylus spp. Genomic DNA amplification of Trichostrongylus colubriformis, Trichostrongylus axei and Trichostrongylus vitrinus was achieved as standard sample using specific primers located in the second internal transcribed spacer (ITSII) of nuclear ribosomal DNA (rDNA). The mentioned method was based on isolation of Trichostrongylus ova from human fecal samples using Willis method, the extraction of ova genomic DNA samples, followed by rDNA ITSII PCR and one-step multiplex RE-PCR using three restriction enzymes of HinfI, DraI, and MseI. The multiplex RE-PCR technique provides a useful tool for discriminating all Trichostrongylus spp., being useful for diagnostic, epidemiological, ecological studies, and control programs. This method is rapid, especially when numerous restriction enzymes are required for species differentiation or identification.

P 182 - Genetic Polymorphism of methylenetetrahydrofolate reductase (MTHFR) C 677 T and A1298C gene and risk of head and neck squamous cell carcinoma-A qualitative analysis

Background and Aim Functional polymorphisms in genes encoding enzymes involved in folate metabolism might modulate head and neck squamous cell carcinoma (HNSCC) risk of which Methylenetetrahydrofolate reductase gene (MTHFR) C677T and A1298C polymorphism is important due to changes in folate levels that can induce disorders in methylation pathway, which results in carcinogenesis. Materials & Methods 25 samples of genomic DNA extracted from whole blood of both HNSCC cases and 25 age matched controls taken & subjected to PCR amplification using specific primers followed by restriction fragment length polymorphism for detection of MTHFR C677T & 1298 C. Interactions with clinical histopathological parameters were also evaluated. Results 1298 AC/CC genotypes are associated with age ≥49 years, tobacco and alcohol (P Conclusion Variables like age ≥49 years, male gender, tobacco and alcohol were associated with MTHFR 1298AC or CC genotypes,indicating that individuals with these variables have higher risk. However further longitudinal studies are required to confirm these as established risk factors. No association between MTHFR C677T polymorphism and HNSCC was possible in this study.

Zebrafish에서 인간 KCNE1 유전자 발현에 관한 연구

This study was aimed to produce a transgenic zebrafish expressing the human KCNE1 gene. Initially, the entire CDS of the human KCNE1 gene was amplified from a human genomic DNA sample by polymerase chain reaction using a primer set engineered with restriction enzyme sites (EcoRⅠ, BamH Ⅰ) at the 5’ end of each primer. The resultant 402 bp KCNE1 amplicon flanked by EcoR1 and BamH1 was obtained and subsequently cloned into a plasmid vector pPB-CMVp-EF1-GreenPuro. The integrity of the cloned CDS sequence was confirmed by DNA sequencing analysis. Next, the recombinant vector containing the human KCNE1 (pPB-CMVp-hKCNE1-EF1-GreenPuro) was introduced into fertilized eggs of zebrafish by microinjection. Successful expression of the recombinant vector in the eggs was confirmed by the expression of the fluorescence protein encoded in the vector. Finally, in order to assure that the stable expression of the human KCNE1 gene occurred in the transgenic animal, RNAs were extracted from the animal and the presence of KCNE1 transcripts was confirmed by RT-PCT as well as DNA sequencing analysis. The study provides a methodology to construct a useful transgenic animal model applicable to the development of diagnostic technologies for gene therapy of LQTS (Long QT Syndrome) as well as tools for cloning of useful genes in fish.

A Multivariate Evaluation of Factors Affecting the Quality of Freshly Frozen Tissue Specimens.

Well-prepared and preserved freshly frozen specimens are indispensable materials for clinical studies. To manage specimen quality and to understand the factors potentially affecting specimen quality during preservation processes, we analyzed the quality of RNA and genomic DNA of various tissues collected between 2002 and 2011 in Linkou Chang Gung Memorial Hospital, Taiwan. During this period, a total of 1059 freshly frozen specimens from eight major cancer categories were examined. It was found that preservation duration, organ origin, and tissue type could all influence the quality of RNA samples. The increased preservation period correlated with decreased RNA quality; the brain, breast, and stomach RNA specimens displayed faster degradation rates than those of other organs, and RNA specimens isolated from tumor tissues were apparently more stable than those of other tissues. These factors could all be used as quality predictors of RNA quality. In contrast, almost all analyses revealed that the genomic DNA samples had good quality, which was not influenced by the aforementioned factors. The results assisted us in determining preservation factors that affect specimen quality, which could provide evidence for improving processes of sample collection and preservation. Furthermore, the results are also useful for researchers to adopt as the evaluation criteria for choosing specimen collection and preservation strategies.

A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population.

BackgroundCongenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required.MethodsWe developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates.ResultsThe test was validated through direct sequencing in a blinded study with 100% concordance.ConclusionsThe results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.

A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.

To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.