PCR-restriction Fragment Length Polymorphism Assay Research Articles

Associations of leptin receptors and miRNA polymorphisms with susceptibility to hypertension.

Leptin receptors (LEPR) are located in the central nervous system and other tissues including adipocytes and endothelial cells, where they play a key role in mediating the effects of leptin. MicroRNA (miR/miRNA)-27a and miR-155 have been shown to play an important role in the regulation of LEPR expression and are differentially expressed in various diseases. Therefore, the present study analyzed potential associations of LEPR deletion/insertion (Del/Ins), miR-27aA>G (rs895819) and miR-155T>A (rs767649) polymorphisms with a predisposition to hypertension (HTN). Genotyping was performed by a PCR-restriction fragment length polymorphism assay. Frequencies of LEPR Del/Ins and miRNA gene polymorphisms in patients diagnosed with HTN (n=232) and randomly selected healthy controls (n=247) were assessed. The present study found that Del/Ins and Ins/Ins genotypes and the Ins allele of the LEPR Del/Ins polymorphism were associated with a decreased risk of HTN compared with controls, whereas the miR-27aA>G rs895819 polymorphism was associated with an increased risk of HTN. Combined genotype and allele analyses for LEPR Del/Ins and two miRNA polymorphisms revealed an association with an increased risk or a decreased risk of HTN. Furthermore, stratification analysis revealed that HTN risk factors were associated with waist circumference (WC) and high-density lipoprotein cholesterol (HDL-C) values in LEPR Del/Ins polymorphism. They were also associated with body mass index, WC, triglyceride and HDL-C values in miR-27aA>G polymorphism. The present study revealed a combined effect of LEPR Del/Ins and miR-27aA>G polymorphisms on the risk of HTN in Koreans, suggesting that these gene polymorphisms could be potential markers for predicting HTN risk.

A Novel 16S rRNA PCR-Restriction Fragment Length Polymorphism Assay to Accurately Distinguish Zoonotic Capnocytophaga canimorsus and C. cynodegmi.

The zoonotic bacteria Capnocytophaga canimorsus and C. cynodegmi, the predominant Capnocytophaga species in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of these Capnocytophaga species using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolated Capnocytophaga spp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using published C. canimorsus and C. cynodegmi 16S rRNA sequences. The results showed that 51% of dogs carried Capnocytophaga spp. Among these, C. cynodegmi (47/98, 48%) was the predominant isolated species along with one strain of C. canimorsus (1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of the C. cynodegmi isolates, which were misidentified as C. canimorsus using previously reported species-specific PCR. Four RFLP types could be classified from all the isolated Capnocytophaga strains. The proposed method demonstrates superior resolution in distinguishing C. cynodegmi (with site-specific polymorphism) from C. canimorsus and especially in distinguishing C. canimorsus from other Capnocytophaga species. After in silico validation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% in C. canimorsus strains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study of Capnocytophaga in small animals and for the rapid diagnosis of human C. canimorsus infections. IMPORTANCE With the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously. Capnocytophaga canimorsus and C. cynodegmi are part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study, C. cynodegmi with site-specific 16S rRNA sequence polymorphisms was erroneously identified as C. canimorsus during the investigation of canine Capnocytophaga by conventional PCR. Consequently, the prevalence of C. canimorsus is incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonotic C. canimorsus from C. cynodegmi. After validation against published Capnocytophaga strains, this novel molecular method had high accuracy and could detect 100% of C. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of human Capnocytophaga infection following exposure to small animals.

TPMT gene polymorphisms (c.238G>C, c.460G>A and c.719A>G) in a healthy Venezuelan population.

Background: The presence of polymorphisms in the TPMT gene is associated with adverse effects in patients treated with standard doses of thiopurine drugs. Scientific evidence recognizes significant ethnic differences in their frequencies and how their early identification can prevent clinical complications. Methods: 150 healthy residents of Aragua, Venezuela were enrolled. The SNPs c.460G>A and c.719A>G were detected by PCR-restriction fragment length polymorphism assay and c.238G>C by allele-specific PCR. Results: All genotype polymorphisms were heterozygous. TPMT*1/*3A, TPMT*1/*3C and TPMT*1/*2 genotypes were found in 4.0, 2.0 and 0.7%, respectively. Conclusion: 6.7% of individuals have an intermediate TPMT activity. These findings support the importance of prior genotyping of TPMT in Venezuelan patients who require thiopurine drug therapy.

Natural variation in the hrpL promoter renders the phytopathogen Pseudomonas syringae pv. actinidiae nonpathogenic.

The genetic basis underlying loss-of-virulence mutations that arise among natural phytopathogen populations is not well documented. In this study, we examined the virulence of 377 isolates of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) that were isolated from 76 kiwifruit orchards suffering from bacterial canker disease. Eighty-four nonpathogenic isolates were identified in 40 orchards. A nonpathogenic isolate G166 was found to be defective in hrpL transcription and the downstream type III secretion system (T3SS)-dependent phenotypes. Comparative genomics and complementary expression assay revealed that a single-base "G" insertion in the hrpL promoter blocks gene transcription by reducing promoter activity. The electrophoretic mobility shift assay showed that the genetic variation impairs σ54 /promoter binding during gene transcription under hrp-inducing conditions, resulting in lower expression of hrpL. A PCR-restriction fragment length polymorphism assay was performed to trace the evolutionary history of this mutation, which revealed the independent onset of genetic variations in natural Psa3 populations. We also found that nonpathogenic variants outperformed virulent Psa3 bacteria for both epiphytic and apoplast colonization of kiwifruit leaves in mixed inoculations. Our study highlights a novel mechanism for loss of virulence in Psa3 and provides insight into bacterial adaptive evolution under natural settings.

Association of CYP3A5*3, CYP3A4*18 & CYP2B6*6 polymorphisms with imatinib treatment outcome in Azerbaijani chronic myeloid leukaemia patients.

Imatinib mesylate (IM) is a reliable first line treatment for chronic myeloid leukaemia (CML). Nevertheless, despite promising results, a considerable proportion of patients develop resistance to the drug. Cytochrome P450 (CYP) enzymes play a crucial role in IM metabolism. Thus, point mutations in CYP genes may modify IM enzyme activity resulting in insufficient treatment response. This investigation was aimed to identify the functional impact of CYP3A5*3, CYP3A4*18 and CYP2B6*6 polymorphisms on the IM response in patients with CML in Azerbaijan. Genotyping of CYP3A5*3, CYP3A4*18 and CYP2B6*6 was performed in 153 patients (102 IM non-responders and 51 IM responders) with CML by the PCR-restriction fragment length polymorphism (RFLP) assays. The odds ratios (ORs) with 95 per cent confidence intervals (CIs) were applied to assess the association between allelic variants and IM therapy outcome. The results were validated by sequencing. The frequency of the CYP3A4*18 allele was considerably lower in the responder's group (97.1 vs. 100%; P=0.036). For CYP3A5*3, the allelic frequency was slightly higher among the IM responders (100 vs. 99.02%) with no significant difference. Although patients heterozygous (TC) for CYP2B6*6 demonstrated a higher risk of acquiring resistance (OR 1.04; 95% CI: 0.492-2.218), differences were not significant (P=0.909). In addition, the homozygous genotype (TT) demonstrated a lower risk of unresponsiveness (OR 0.72; 95% CI: 0.283-1.836), but associations were not significant (P=0.491). Our results demonstrated that CYP3A4*18 was significantly associated with IM treatment response in patients with CML in Azerbaijan, whereas rather common CYP3A5*3 was identified to have no such association.

Population structure, molecular characterization, and phylogenetic analysis of Fasciola gigantica from two locations in Uganda.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Association of polymorphisms of VEGF and VEGFR1 pathways related genes and risk of pre-eclampsia

To assess the association of single nucleotide polymorphisms (SNPs) of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 1 (VEGFR1) pathways-related genes and the risk of pre-eclampsia. In total 178 pregnant women with pre-eclampsia (case group) and 100 healthy pregnant women (control group) during the third trimester were enrolled. The SNPs of VEGF rs3025039, rs2010963 and VEGFR1 rs3812867, rs55875014 and rs722503 loci were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The levels of serum VEGF and sVEGFR1 were also determined. And their association with pre-eclampsia was analyzed. The systolic blood pressure, diastolic blood pressure and sVEGFR1 of the case group were significantly higher than those of the control group, while the VEGF level was significantly lower than that in the control group (P<0.05). Allelic frequencies of the VEGF (rs3025039, rs2010963) and VEGFR1 (rs3812867, rs55875014, rs722503) have fit the Hardy-Weinberg equilibrium (P>0.05). The frequency of T allele of VEGF at rs3025039 locus in the case group was higher than that in the control group (P<0.05). There were significant differences in VEGF at rs3025039 locus under dominant and co-dominant models in case group (P<0.05). Compared with those with CC, the risk was higher in patients with CT or TT genotypes (P<0.05). The systolic and diastolic blood pressure and sVEGFR1 in pre-eclampsia pregnant women with CT or TT genotypes were significantly higher than those with the CC genotype, while their VEGF level was significantly lower (P<0.05). No significant difference was found in allelic frequencies of other four loci between the two groups (P>0.05). Polymorphisms of rs3025039 locus of VEGF gene is associated with the occurrence of pre-eclampsia. The variant at this locus may affect the activity of VEGF and influence the development of pre-eclampsia.

Molecular detection of fluoroquinolone-resistant Neisseria meningitidis by using mismatched PCR-restriction fragment length polymorphism technique.

Ciprofloxacin (CIP) is a commonly used antibiotic for meningococcal chemoprophylaxis, and the mutations in the quinolone resistance-determining region of gyrA are associated with CIP-resistant Neisseria meningitidis. Here, we established a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect a mutation at codon 91 of gyrA, followed by high-level CIP-resistant meningococci. We designed PCR-RFLP primers to detect the T91I mutation in gyrA by introducing an artificial AciI cleavage site. This assay was performed using 26 N. meningitidis strains whose gyrA sequences have been characterized. The amplified 160 bp PCR product from gyrA was digested into three fragments (80, 66, and 14 bp) when there was no mutation, or two fragments (146 and 14 bp) when there was a mutation at codon 91. A correlation was observed between the mismatched PCR-RFLP assay and gyrA sequencing. This rapid, simple, and accurate assay has the potential to detect CIP-resistant N. meningitidis in clinical microbiology laboratories, contributing to the appropriate antibiotic selection for meningococcal chemoprophylaxis, will help maintain an effective treatment for close contacts of IMD patients, and prevent the spread of CIP-resistant N. meningitidis.

Tumor Necrosis Factor Receptor II Gene Polymorphism and Association with Plasmodium falciparum Malaria in Badagry, Lagos, Nigeria

Host genetic factors, host-parasite interactions, parasite genetic diversity, and/or environmental influences have been implicated in the molecular basis of susceptibility and resistance to Plasmodium parasite infections. Host Tumor Necrosis Factor Receptor II (TNFR II), a 75/80kDa protein encoded by the tnfr II gene has been identified as an adhesion protein for Plasmodium falciparum parasite. Tnfr II gene exhibits functional polymorphism on exon 6 among Caucasians with a potential effect on the ligand-binding function of TNFR II. This study aimed to investigate tnfr II gene polymorphisms on exon 6 and its relationship to Plasmodium falciparum malaria in Badagry, Lagos Nigeria. Genotyping was done using PCR-Restriction fragment length polymorphism assay. A total of 68 blood samples comprising of 49 controls and 19 malaria cases were genotyped. Result revealed both the wild-type (T587) and a mutant-type (T→587G) genotypes of tnfr II. Generally, the analysis showed that the variant-type had a lower prevalence rate (11.76%) than the wild-type (88.24%). The frequencies of wild-type were highest in the controls (85.70%) while the frequencies of the mutant-type were lower in the malaria cases (5.30%). However, the Chi-square test showed no statistical significant difference (p value = 0.30) between the distribution of the genotypes in malaria cases and controls. This study indicates that polymorphism on exon 6 of tnfr II gene was not associated with susceptibility or resistance to malaria infection in Badagry, Nigeria. Further researches with a larger sample size are needed to clarify this subject.

Anopheles gambiae strain (Ag55) cultured cells originated from Anopheles coluzzii and are phagocytic with hemocyte-like gene expression.

Anopheles gambiae and Anopheles coluzzii are closely related species that are predominant vectors of malaria in Africa. Recently, A. gambiae form M was renamed A. coluzzii and we now conclude on the basis of a diagnostic PCR-restriction fragment length polymorphism assay that Ag55 cells were derived from A. coluzzii. We established an Ag55 cell transcriptome, and KEGG pathway analysis showed that Ag55 cells are enriched in phagosome pathway transcripts. The Ag55 transcriptome has an abundance of specific transcripts characteristic of mosquito hemocytes. Functional E. coli bioparticle uptake experiments visualized by fluorescence microscopy and confocal microscopy and quantified by flow cytometry establish the phagocytic competence of Ag55 cells. Results from this investigation of Ag55 cell properties will guide researchers in the use and engineering of the Ag55 cell line to better enable investigations of Plasmodium, other microbes, and insecticidal toxins.

Phlebotomine Sand Flies in Southern Thailand: Entomological Survey, Identification of Blood Meals and Molecular Detection of Trypanosoma spp.

Simple SummaryPhlebotomine sand flies (Diptera: Psychodidae) are hematophagous insects, and many species serve as vectors of various human and animal pathogens, including Leishmania and Trypanosoma protozoa. In Thailand, the first case of autochthonous leishmaniasis was reported 62 years ago. At present, the number of human cases is increasing in different regions of the country, but most cases are reported from southern Thailand. Therefore, we studied the potential transmission of Leishmania and Trypanosoma by sand flies in three provinces of southern Thailand, and analyzed blood sources of engorged sand fly females. We detected Trypanosoma sp. DNA in Sergentomyia barraudi, S. indica, S. khawi and Idiophlebotomus asperulus but no Leishmania spp. DNA. Moreover, bloodmeal analysis revealed that Trypanopsoma-positive females of S. barraudi and Sergentomyia sp. fed on dogs and humans, respectively. The results of this study contribute to the knowledge of leishmaniasis and trypanosomiasis presence and sand fly feeding behavior in southern Thailand.An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.

Effect of major CYP2C19 genetic polymorphisms on Helicobacter pylori eradication based on different treatment regimens.

Helicobacter pylori (H. pylori) infection is a global issue. Its eradication in affected individuals is important to prevent several further complications that may occur if left untreated. Proton pump inhibitors (PPIs) serve an important role in the eradication regimens of H. pylori. PPIs are metabolized primarily through the CYP2C19 enzyme in the liver. Inter-individual variation in the response to eradication treatment may partly be due to variations in the metabolism of PPIs. The aim of this study was to determine whether there was any association between CYP2C19 genetic polymorphisms and the response to eradication therapy amongst Jordanians infected with H. pylori receiving lansoprazole-based regimens. The present study was approved by the Institutional Review Board of The University of Jordan Hospital. A total of 141 patients infected with H. pylori were genotyped for the polymorphisms CYP2C19*2 and CYP2C19*17 using the PCR-restriction fragment length polymorphism assay method. Patients received lansoprazole-based triple or sequential therapy. The assessment of eradication was performed using either a H. pylori stool antigen test or from feedback from patients regarding their improvement. Eradication rates were 84.6% and 64.5% in the intermediate-metabolizer and extensive-metabolizer group, respectively. This difference was not statistically significant. Moreover, no significant association was found between the carriers of the CYP2C19*17 polymorphism and the response to eradication therapy. These findings suggest that there was no significant association between the CYP2C19 genotype and the response to eradication therapy amongst Jordanians infected with H. pylori.

Tumor necrosis factor-alpha-induced protein 3 gene polymorphism and systemic lupus erythematosus susceptibility in Egyptian population

Objectives To clarify the association of tumor necrosis factor-alpha-induced protein 3 (TNFAIP3) rs5029939 polymorphism with systemic lupus erythematosus (SLE) susceptibility and pathogenesis in the Egyptian population. Background TNFAIP3 is a single nucleotide polymorphisms coding for the ubiquitin-modifying enzyme, associated with autoimmune diseases like SLE. Patients and methods A case–control study included 80 patients with SLE as well as 80 controls. Clinical assessment, renal biopsy, and full laboratory investigations were done. DNA samples were tested for TNFAIP3 using PCR-restriction fragment length polymorphism assay. Results TNFAIP3 (rs5029939 C > G) genotype distribution showed no statistically significant difference between patients with SLE and controls (P = 0.226); odds ratio (95% confidence interval) of C/C, C/G, and G/G genotypes was 0.89 (0.68–1.04), 1.38 (0.92–1.82), and 1.02 (0.71–1.1), respectively. The most frequent allele in the two groups was C allele in group I and group II (85 and 90%, respectively). Conclusions The studied sample of the Egyptian population carrying TNFAIP3 polymorphism has no susceptibility to developing SLE. Further studies are still needed to verify these results, including a larger number of patients to illuminate the potential role of the TNFAIP3 (rs5029939 C > G) gene polymorphism in SLE pathogenesis in Egypt. Further studies are recommended to detect other polymorphisms in the TNFAIP3 gene like rs2230926 in the Egyptian population, investigating the specific ethnic patients' contribution.

PCR-RFLP assay confirms the existence of different mitochondrial lineages of Taenia hydatigena including a possible geographically restricted group.

Taenia hydatigena is a widespread tapeworm of canids (primarily dogs) that causes cysticercosis in ruminants (domestic and wild) and manifests as depression and weakness secondary to various hepatic damages and sometimes mortality in young animals, although, commonly encountered cases are asymptomatic. In most taeniids, genetic polymorphism has been found to impact host preferences, distribution, disease epidemiology and management. Recently, we identified two main mitochondrial lineages of T.hydatigena in China, and here, we examined the mitochondrial nad4-nad5 genes of T.hydatigena from China, Nigeria, Pakistan and Sudan to assess the intraspecies variation of isolates from these countries and also the distribution of the distinct mitochondrial groups. In addition to China, haplogroup B variant was found in Pakistan, while haplogroup A demonstrated a widespread distribution. We then designed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay using XmiI (AccI) and RsaI (AfaI) restriction enzymes to differentiate members of both haplogroups. This result provides more molecular evidence supporting the existence of distinct mitochondrial variants of T.hydatigena. The epidemiological significance of these different mitochondrial groups remains to be explored further. The current PCR-RFLP assay offers a useful molecular approach for investigating the genetic population structure of T.hydatigena in enzootic regions and in identifying/discriminating the different mitochondrial groups (haplogroups A and B).

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods

Ethnopharmacological relevanceThe roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. Aim of this studyThis study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. Materials and methodsThe sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. ResultsThe plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. ConclusionThe nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

BackgroundThe prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs.ResultFirst, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays.ConclusionIn this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P&amp;gt;0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

Continued Upward Trend in Non-Tuberculous Mycobacteria Isolation over 13 Years in a Tertiary Care Hospital in Korea.

PurposeDespite decreased prevalence of tuberculosis, the incidence of the diseases associated with nontuberculous mycobacteria (NTM) has been increasing in South Korea and around the world. The present retrospective study was conducted to determine longitudinal changes in the epidemiology and distribution of NTM over 13 years at a tertiary care hospital in Korea.Materials and MethodsWe retrospectively analyzed data on Mycobacterium species over 13 years (January 2007 to December 2019) by utilizing the laboratory information system. Mycobacterium species were identified using biochemical tests and PCR-restriction fragment length polymorphism and Mycobacteria GenoBlot assays.ResultsAfter excluding duplicates from the initial pool of 17996 mycobacterial isolates, 7674 strains were analyzed and 2984 (38.9%) NTM were isolated. The proportion of NTM continuously increased over the 13-year period, from 17.0% in 2007 to 57.5% in 2019. Among the NTM isolates, the most common species were Mycobacterium intracellulare (50.6%), M. avium (18.3%), M. fortuitumcomplex (4.9%), M. abscessus (4.5%), M. gordonae (3.3%), M. kansasii (1.1%), M. chelonae (1.0%), and M. massiliense (0.9%). In patients over the age of 70 years, the proportion of NTM among the isolates increased from 26.6% in 2007 to 62.0% in 2019, and that of M. intracellulare isolates among the NTM increased from 13.9% (11/79) in 2007 to 37.4% (175/468) in 2019.ConclusionThe number of NTM isolates continuously increased over the study period, and the increase in the proportion of M.intracellulare in patients aged over 70 years was notable.

High levels of leptin and non-high molecular weight-adiponectin in patients with colorectal cancer: Association with chemotherapy and common genetic polymorphisms.

Colorectal cancer (CRC) is the third most frequently diagnosed cancer worldwide. Leptin and adiponectin are hormones produced by adipose tissues, which exhibit opposing effects on tumor growth. Leptin promotes tumor development and metastasis, whereas adiponectin attenuates this. The aim of the present study was to assess the possible association between leptin and adiponectin [both high molecular weight (HMW) and non-HMW factions] levels with CRC, CRC response to chemotherapy, and to study the relationship between LEPR (rs6588147), ADIPO (rs266729), LEP (rs2167270), and ADIPO (rs822369) polymorphisms and CRC. A total of 32 blood samples collected from CRC patients were analyzed to identify the serum levels of leptin and adiponectin, and the presence of CRC related polymorphisms. A total of 25 healthy subjects were recruited in the control group. Serum levels of leptin and adiponectin were detected using ELISA whereas DNA from patients and controls was amplified and analyzed using PCR-restriction fragment length polymorphism assay. The results showed that the levels of leptin and non-HMW adiponectin were significantly higher in CRC patients compared with the controls (P<0.05). In addition, HMW adiponectin was significantly higher in patients receiving chemotherapy. The association between LEPR (rs6588147), ADIPO (rs266729), LEP (rs2167270) and ADIPO (rs822369) polymorphisms and CRC was not significant (P>0.05). In conclusion, higher leptin and non-HMW adiponectin levels may be associated with increased CRC. Chemotherapy may positively influence the levels of HMW adiponectin. No association between LEPR (rs6588147), ADIPO (rs266729), LEP (rs2167270) and ADIPO (rs822369) polymorphisms with CRC was found.

Genotyping 5,10-methylenetetrahydrofolate reductase for patients with coronary artery disease in Southern Taiwan

Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease (CAD). Individuals with homozygous MTHFR (5,10-Methylenetetrahydrofolate reductase) mutation have significantly elevated plasma homocysteine levels. Two common mutations in MTHFR (MTHFR 677C→T and MTHFR 1298A→C) may represent an important genetic risk factor in vascular disease. The aim of this research was to investigate these two point mutations of MTHFR for CAD patients and non-CAD controls in Southern Taiwan by PCR-restriction fragment length polymorphism assay. We found that the distribution of the homozygous mutation for MTHFR677 was higher in 54 CAD patients than in 55 non-CAD subjects (11.1% versus 3.6%, P = 0.275). On the other hand, the frequency of MTHFR 1298CC in CAD patients was similar to that of control subjects (P = 1.000). This may suggest there is a trend toward an increased risk of coronary heart disease for individuals that have a 677TT genotype in the Taiwanese population. Finally, the frequency of homozygosity for the two MTHFR common mutations was still low in CAD cases. There may not be an appreciable association of MTHFR mutation to cardiovascular disease in Taiwan. Perhaps a further study including assessment of plasma concentrations of homocysteine, folate and vitamin B12 would be needed to better clarify the relationship between MTHFR polymorphisms and the occurrence of vascular disease.