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Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Hussain H, Chong NF

Biomed Res Int, 2016;2016:8041532.
PMID: 27995143

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

Matched MeSH terms: DNA/genetics*
Fulltext Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins

Fischer H, Tschachler E, Eckhart L

Apoptosis, 2020 08;25(7-8):474-480.
PMID: 32533513 DOI: 10.1007/s10495-020-01614-4

The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.

Matched MeSH terms: DNA/genetics*
Cytotoxicity and immunological responses following oral vaccination of nanoencapsulated avian influenza virus H5 DNA vaccine with green synthesis silver nanoparticles

Jazayeri SD, Ideris A, Zakaria Z, Shameli K, Moeini H, Omar AR

J Control Release, 2012 Jul 10;161(1):116-23.
PMID: 22549012 DOI: 10.1016/j.jconrel.2012.04.015

DNA formulations provide the basis for safe and cost effective vaccine. Low efficiency is often observed in the delivery of DNA vaccines. In order to assess a new strategy for oral DNA vaccine formulation and delivery, plasmid encoding hemagglutinin (HA) gene of avian influenza virus, A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1/H5) was formulated using green synthesis of sliver nanoparticles (AgNP) with polyethylene glycol (PEG). AgNP were successfully synthesized uniformly dispersed with size in the range of 4 to 18 nm with an average size of 11 nm. Cytotoxicity of the prepared AgNP was investigated in vitro and in vivo using MCF-7 cells and cytokine expression, respectively. At the concentration of -5 log₁₀AgNP, no cytotoxic effects were detected in MCF-7 cells with 9.5% cell death compared to the control. One-day-old specific pathogen-free (SPF) chicks immunized once by oral gavage with 10 μl of pcDNA3.1/H5 (200 ng/ml) nanoencapsulated with 40 μl AgNP (3.7×10⁻² μg of Ag) showed no clinical manifestations. PCR successfully detect the AgNP/H5 plasmid from the duodenum of the inoculated chicken as early as 1h post-immunization. Immunization of chickens with AgNP/H5 enhanced both pro inflammatory and Th1-like expressions, although no significant differences were recorded in the chickens inoculated with AgNP, AgNP/pcDNA3.1 and the control. In addition, serum samples collected from immunized chickens with AgNP/H5 showed rapidly increasing antibody against H5 on day 14 after immunization. The highest average antibody titres were detected on day 35 post-immunization at 51.2±7.5. AgNP/H5 also elicited both CD4+ and CD8+ T cells in the immunized chickens as early as day 14 after immunization, at 7.5±2.0 and 20±1.9 percentage, respectively. Hence, single oral administrations of AgNP/H5 led to induce both the antibody and cell-mediated immune responses as well as enhanced cytokine production.

Matched MeSH terms: Vaccines, DNA/genetics
DNA Hairpins and Stabilization of Gold Nanoparticles: Effect of Stem Length and Toehold Composition

Ooi JSY, Lim CR, Hua CX, Ng JF, New SY

Langmuir, 2023 Oct 31;39(43):15200-15207.
PMID: 37851548 DOI: 10.1021/acs.langmuir.3c01748

This study investigates the effect of DNA hairpins on the stabilization of gold nanoparticles (AuNPs) against salt-induced aggregation (SIA) in label-free colorimetric biosensors. AuNPs were incubated with DNA hairpins of varying stem lengths and toehold sequences, followed by the addition of NaCl, before being subjected to ultraviolet-visible (UV-vis) measurement. Results showed that hairpins with longer stems generally provide better stabilization of AuNPs (18-bp >14-bp >10-bp). No improvement was observed for 14- and 18-bp hairpins with a toehold beyond 8A, which may be attributed to saturated adsorption of hairpins on the gold surface. For 14-bp hairpins with an 8-mer homopolymeric toehold, we observed a stabilization trend of A > C > G > T, similar to the reported trend of ssDNA. For variants containing ≥50% adenine as terminal bases, introducing cytosine or guanine as preceding bases could also result in strong stabilization. As the proportion of adenine decreases, variants with guanine or thymine provide less protection against SIA, especially for guanine-rich hairpins (≥6G) that could form G-quadruplexes. Such findings could serve as guidelines for researchers to design suitable DNA hairpins for label-free AuNP-based biosensors.

Matched MeSH terms: DNA/genetics
DNA Switch: Toehold-Mediated DNA Isothermal Amplification for Dengue Serotyping

Chan SK, Kuzuya A, Choong YS, Lim TS

SLAS Discov, 2019 01;24(1):68-76.
PMID: 30063871 DOI: 10.1177/2472555218791743

The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.

Matched MeSH terms: DNA/genetics*
DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods

Ayoib A, Hashim U, Gopinath SCB, Md Arshad MK

Appl Microbiol Biotechnol, 2017 Nov;101(22):8077-8088.
PMID: 28942548 DOI: 10.1007/s00253-017-8493-0

This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

Matched MeSH terms: DNA/genetics
DNA-RNA complementation on silicon wafer for thyroid cancer determination

Gopinath SCB, Xuan S

Biotechnol Appl Biochem, 2021 Jun;68(3):554-559.
PMID: 32460382 DOI: 10.1002/bab.1961

One of the current issues with thyroid tumor is early diagnosis as it makes the higher possibility of curing. This research was focused to detect and quantify the level of specific target sequence complementation of miR-222 with capture DNA sequence on interdigitated electrode (IDE) sensor. The aluminum electrode with the gap and finger sizes of 10 µm was fabricated on silicon wafer, further the surface was amine-functionalized for accommodating carboxylated-DNA probe. With DNA-target RNA complementation, the detection limit was attained to be 1 fM as estimated by a linear regression analysis [y = 1.5325x - 2.1171 R² = 0.9065] and the sensitivity was at the similar level. Current responses were higher by increasing the target RNA sequence concentrations. Control experiments with mismatched/noncomplementary sequences were failed to complement the capture DNA sequence immobilized on IDE, indicating the specific target validation. This research helps diagnosing and identifying the progression with thyroid tumor and miRNA being a potential "marker" in atypia diagnosis.

Matched MeSH terms: DNA/genetics*
Fulltext DNA-based taxonomy of a mangrove-associated community of fishes in Southeast Asia

Zainal Abidin DH, Mohd Nor SA, Lavoué S, A Rahim M, Jamaludin NA, Mohammed Akib NA

Sci Rep, 2021 Sep 07;11(1):17800.
PMID: 34493747 DOI: 10.1038/s41598-021-97324-1

The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.

Matched MeSH terms: DNA/genetics
DNA/Nano based advanced genetic detection tools for authentication of species: Strategies, prospects and limitations

Khalil I, Hashem A, Nath AR, Muhd Julkapli N, Yehye WA, Basirun WJ

Mol Cell Probes, 2021 10;59:101758.
PMID: 34252563 DOI: 10.1016/j.mcp.2021.101758

Authentication, detection and quantification of ingredients, and adulterants in food, meat, and meat products are of high importance these days. The conventional techniques for the detection of meat species based on lipid, protein and DNA biomarkers are facing challenges due to the poor selectivity, sensitivity and unsuitability for processed food products or complex food matrices. On the other hand, DNA based molecular techniques and nanoparticle based DNA biosensing strategies are gathering huge attention from the scientific communities, researchers and are considered as one of the best alternatives to the conventional strategies. Though nucleic acid based molecular techniques such as PCR and DNA sequencing are getting greater successes in species detection, they are still facing problems from its point-of-care applications. In this context, nanoparticle based DNA biosensors have gathered successes in some extent but not to a satisfactory stage to mark with. In recent years, many articles have been published in the area of progressive nucleic acid-based technologies, however there are very few review articles on DNA nanobiosensors in food science and technology. In this review, we present the fundamentals of DNA based molecular techniques such as PCR, DNA sequencing and their applications in food science. Moreover, the in-depth discussions of different DNA biosensing strategies or more specifically electrochemical and optical DNA nanobiosensors are presented. In addition, the significance of DNA nanobiosensors over other advanced detection technologies is discussed, focusing on the deficiencies, advantages as well as current challenges to ameliorate with the direction for future development.

Matched MeSH terms: DNA/genetics
Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis

Jarolim P, Palek J, Amato D, Hassan K, Sapak P, Nurse GT, et al.

Proc Natl Acad Sci U S A, 1991 Dec 15;88(24):11022-6.
PMID: 1722314

Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.

Matched MeSH terms: DNA/genetics
Developing critical thinking in STEM education through inquiry-based writing in the laboratory classroom

Jeon AJ, Kellogg D, Khan MA, Tucker-Kellogg G

Biochem Mol Biol Educ, 2021 01;49(1):140-150.
PMID: 32746505 DOI: 10.1002/bmb.21414

Laboratory pedagogy is moving away from step-by-step instructions and toward inquiry-based learning, but only now developing methods for integrating inquiry-based writing (IBW) practices into the laboratory course. Based on an earlier proposal (Science 2011;332:919), we designed and implemented an IBW sequence in a university bioinformatics course. We automatically generated unique, double-blinded, biologically plausible DNA sequences for each student. After guided instruction, students investigated sequences independently and responded through IBW writing assignments. IBW assignments were structured as condensed versions of a scientific research article, and because the sequences were double blinded, they were also assessed as authentic science and evaluated on clarity and persuasiveness. We piloted the approach in a seven-day workshop (35 students) at Perdana University in Malaysia. We observed dramatically improved student engagement and indirect evidence of improved learning outcomes over a similar workshop without IBW. Based on student feedback, initial discomfort with the writing component abated in favor of an overall positive response and increasing comfort with the high demands of student writing. Similarly, encouraging results were found in a semester length undergraduate module at the National University of Singapore (155 students).

Matched MeSH terms: DNA/genetics
Fulltext Development and immunogenic potentials of chitosan-saponin encapsulated DNA vaccine against avian infectious bronchitis coronavirus

Bande F, Arshad SS, Bejo MH, Omar AR, Moeini H, Khadkodaei S, et al.

Microb Pathog, 2020 Dec;149:104560.
PMID: 33068733 DOI: 10.1016/j.micpath.2020.104560

Infectious Bronchitis (IB) is an economically important avian disease that considerably threatens the global poultry industry. This is partly, as a result of its negative consequences on egg production, weight gain as well as mortality rate.The disease is caused by a constantly evolving avian infectious bronchitis virus whose isolates are classified into several serotypes and genotypes that demonstrate little or no cross protection. In order to curb the menace of the disease therefore, broad based vaccines are urgently needed. The aim of this study was to develop a recombinant DNA vaccine candidate for improved protection of avian infectious bronchitis in poultry. Using bioinformatics and molecular cloning procedures, sets of monovalent and bivalent DNA vaccine constructs were developed based on the S1 glycoprotein from classical and variants IBV strains namely, M41 and CR88 respectively. The candidate vaccine was then encapsulated with a chitosan and saponin formulated nanoparticle for enhanced immunogenicity and protective capacity. RT-PCR assay and IFAT were used to confirm the transcriptional and translational expression of the encoded proteins respectively, while ELISA and Flow-cytometry were used to evaluate the immunogenicity of the candidate vaccine following immunization of various SPF chicken groups (A-F). Furthermore, histopathological changes and virus shedding were determined by quantitative realtime PCR assay and lesion scoring procedure respectively following challenge of various subgroups with respective wild-type IBV viruses. Results obtained from this study showed that, groups vaccinated with a bivalent DNA vaccine construct (pBudCR88-S1/M41-S1) had a significant increase in anti-IBV antibodies, CD3+ and CD8+ T-cells responses as compared to non-vaccinated groups. Likewise, the bivalent vaccine candidate significantly decreased the oropharyngeal and cloacal virus shedding (p < 0.05) compared to non-vaccinated control. Chickens immunized with the bivalent vaccine also exhibited milder clinical signs as well as low tracheal and kidney lesion scores following virus challenge when compared to control groups. Collectively, the present study demonstrated that bivalent DNA vaccine co-expressing dual S1 glycoprotein induced strong immune responses capable of protecting chickens against infection with both M41 and CR88 IBV strains. Moreso, it was evident that encapsulation of the vaccine with chitosan-saponin nanoparticle further enhanced immune responses and abrogates the need for multiple booster administration of vaccine. Therefore, the bivalent DNA vaccine could serve as efficient and effective alternative strategy for the control of IB in poultry.

Matched MeSH terms: Vaccines, DNA/genetics
Fulltext Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery

Bahadoran A, Moeini H, Bejo MH, Hussein MZ, Omar AR

J Pharm Pharm Sci, 2016 Jul-Sep;19(3):325-338.
PMID: 27806247 DOI: 10.18433/J3G31Q

PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed.

METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry.

RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05).

CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Matched MeSH terms: Vaccines, DNA/genetics
Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV

Moeini H, Omar AR, Rahim RA, Yusoff K

Comp Immunol Microbiol Infect Dis, 2011 May;34(3):227-36.
PMID: 21146874 DOI: 10.1016/j.cimid.2010.11.006

In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.

Matched MeSH terms: Vaccines, DNA/genetics
Fulltext Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Nordin N, Yusof NA, Radu S, Hushiarian R

J Vis Exp, 2018 06 03.
PMID: 29912194 DOI: 10.3791/56585

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

Matched MeSH terms: DNA/genetics*
Development of single-locus DNA microsatellite markers using 5'anchored ISSR-PCR method for the mangrove horseshoe crab, Carcinoscorpius rotundicauda (Latreille, 1802) in Peninsular Malaysia

Adibah AB, Ling LP, Tan SG, Faridah QZ, Christianus A

Mol Biol Rep, 2012 Apr;39(4):3815-20.
PMID: 21744263 DOI: 10.1007/s11033-011-1159-6

Horseshoe crabs are said to be declining worldwide. However, there is still no published report on the status of horseshoe crabs in Malaysia. Thus, we report here eight informative microsatellite markers that were developed using the 5'-anchored ISSR-PCR enrichment procedure to diagnose the population genetic structure of the mangrove horseshoe crab, Carcinoscorpius rotundicauda from Peninsular Malaysia. This set of markers was tested on 127 samples and showed polymorphism in this species. Hence they should be useful in future essential population genetic studies of these living fossils in the Southeast Asian region.

Matched MeSH terms: DNA/genetics*
Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations

Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS, et al.

Pharmacogenetics, 2002 Aug;12(6):437-50.
PMID: 12172212

The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.

Matched MeSH terms: DNA/genetics
Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia

Iwai K, Hirono A, Matsuoka H, Kawamoto F, Horie T, Lin K, et al.

Hum Genet, 2001 Jun;108(6):445-9.
PMID: 11499668

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.

Matched MeSH terms: DNA/genetics
Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder

Asing, Ali E, Hamid SB, Hossain M, Ahamad MN, Hossain SM, et al.

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2016 Nov;33(11):1643-1659.
PMID: 27643977

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.

Matched MeSH terms: DNA/genetics
Efficacy of Rosuvastatin in Children With Homozygous Familial Hypercholesterolemia and Association With Underlying Genetic Mutations

Stein EA, Dann EJ, Wiegman A, Skovby F, Gaudet D, Sokal E, et al.

J Am Coll Cardiol, 2017 Aug 29;70(9):1162-1170.
PMID: 28838366 DOI: 10.1016/j.jacc.2017.06.058

BACKGROUND: Homozygous familial hypercholesterolemia (HoFH), a rare genetic disorder, is characterized by extremely elevated levels of low-density lipoprotein cholesterol (LDL-C) and accelerated atherosclerotic cardiovascular disease. Statin treatment starts at diagnosis, but no statin has been formally evaluated in, or approved for, HoFH children.
OBJECTIVES: The authors sought to assess the LDL-C efficacy of rosuvastatin versus placebo in HoFH children, and the relationship with underlying genetic mutations.
METHODS: This was a randomized, double-blind, 12-week, crossover study of rosuvastatin 20 mg versus placebo, followed by 12 weeks of open-label rosuvastatin. Patients discontinued all lipid-lowering treatment except ezetimibe and/or apheresis. Clinical and laboratory assessments were performed every 6 weeks. The relationship between LDL-C response and genetic mutations was assessed by adding children and adults from a prior HoFH rosuvastatin trial.
RESULTS: Twenty patients were screened, 14 randomized, and 13 completed the study. The mean age was 10.9 years; 8 patients were on ezetimibe and 7 on apheresis. Mean LDL-C was 481 mg/dl (range: 229 to 742 mg/dl) on placebo and 396 mg/dl (range: 130 to 700 mg/dl) on rosuvastatin, producing a mean 85.4 mg/dl (22.3%) difference (p = 0.005). Efficacy was similar regardless of age or use of ezetimibe or apheresis, and was maintained for 12 weeks. Adverse events were few and not serious. Patients with 2 defective versus 2 negative LDL receptor mutations had mean LDL-C reductions of 23.5% (p = 0.0044) and 14% (p = 0.038), respectively.
CONCLUSIONS: This first-ever pediatric HoFH statin trial demonstrated safe and effective LDL-C reduction with rosuvastatin 20 mg alone or added to ezetimibe and/or apheresis. The LDL-C response in children and adults was related to underlying genetic mutations. (A Study to Evaluate the Efficacy and Safety of Rosuvastatin in Children and Adolescents With Homozygous Familial Hypercholesterolemia [HYDRA]; NCT02226198).

Matched MeSH terms: DNA/genetics*

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