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Universal mini COI barcode for the identification of fish species in processed products

Sultana S, Ali ME, Hossain MAM, Asing, Naquiah N, Zaidul ISM

Food Res Int, 2018 03;105:19-28.
PMID: 29433207 DOI: 10.1016/j.foodres.2017.10.065

Species substitution, the use of a low value fish in place of a high value fish, is the biggest problem in international trade and the leading cause of fraud in the fisheries arena sector. Current DNA barcoding systems have partly solved this problem but also failed in many instances to amplify PCR targets from highly processed products because of the degradation of a longer barcode marker (~650bp). In the present study, a novel mini barcode marker (295bp) was developed to discriminate fish species in raw and processed states forms. The barcode primers were cross-tested against 33 fish species and 15 other animal species and found to be universal for all the tested fish varieties. When 20 commercial fish products of five different categories were screened, all commercial fish sample yielded positive bands for the novel fish barcode. PCR product was sequenced to retrieve the species IDs that reflected 55% (11/20) of Malaysian fish products were mislabeled.

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
Fulltext Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines

Asing, Ali ME, Abd Hamid SB, Hossain MA, Mustafa S, Kader MA, et al.

PLoS One, 2016;11(10):e0163436.
PMID: 27716792 DOI: 10.1371/journal.pone.0163436

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Matched MeSH terms: 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 Improving the potency of DNA vaccine against chicken anemia virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) type-1 VP22 protein

Moeini H, Omar AR, Rahim RA, Yusoff K

Virol J, 2011;8:119.
PMID: 21401953 DOI: 10.1186/1743-422X-8-119

Studies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV).

Matched MeSH terms: Vaccines, DNA/genetics
Improved protection from velogenic Newcastle disease virus challenge following multiple immunizations with plasmid DNA encoding for F and HN genes

Loke CF, Omar AR, Raha AR, Yusoff K

Vet Immunol Immunopathol, 2005 Jul 15;106(3-4):259-67.
PMID: 15963824

Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.

Matched MeSH terms: Vaccines, DNA/genetics
Apolipoprotein E genotyping in the Malay, Chinese and Indian ethnic groups in Malaysia - a study on the distribution of the different apoE alleles and genotypes

Seet WT, Mary Anne TJ, Yen TS

Clin Chim Acta, 2004 Feb;340(1-2):201-5.
PMID: 14734213 DOI: 10.1016/j.cccn.2003.11.001

BACKGROUND: Apolipoprotein E (apoE) is encoded by a polymorphic gene located on chromosome 19. The three common apoE alleles are epsilon2, epsilon3 and epsilon4. We studied the frequencies of the apoE alleles and genotypes in the three ethnic groups-Malay, Chinese and Indian-in Malaysia using DNA amplification followed by agarose gel electrophoresis.
METHODS: EDTA blood was collected and DNA was extracted using proteinase K-SDS digestion and purified by phenol-chloroform extraction. The apoE gene sequence was amplified using the PCR and apoE genotyping was performed by restriction enzyme digestion with HhaI.
RESULTS: Genotyping of the apoE gene produces six genotypes-E2/E2, E2/E3, E3/E3, E2/E4, E3/E4 and E4/E4. The most common apoE genotype in the Malays, Chinese and Indians studied was E3/E3, thus the most common apoE allele was epsilon3. The three common apoE genotypes were E3/E3 followed by E3/E4 and E2/E3, except in the Indians where E2/E3 was not detected. The three apoE alleles were confirmed in the Malays, Chinese and Indians except for the epsilon2 allele which was absent in the Indians.
CONCLUSION: The combined frequency of the apoE alleles in the Malays, Chinese and Indians was 0.058, 0.829 and 0.114 for epsilon2, epsilon3 and epsilon4, respectively.

Matched MeSH terms: DNA/genetics
Fulltext Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia

Okuma HS, Yoshida H, Kobayashi Y, Arakaki M, Mizoguchi C, Inagaki L, et al.

Cancer Sci, 2023 Jun;114(6):2664-2673.
PMID: 36919757 DOI: 10.1111/cas.15790

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p

Matched MeSH terms: DNA/genetics
Fulltext Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications

Siew WS, Tang YQ, Kong CK, Goh BH, Zacchigna S, Dua K, et al.

Int J Mol Sci, 2021 Aug 05;22(16).
PMID: 34445123 DOI: 10.3390/ijms22168422

Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.

Matched MeSH terms: DNA/genetics
Fulltext Characterisation of beta-globin gene mutations in Malaysian children: a strategy for the control of beta-thalassaemia in a developing country

Thong MK, Tan JA, Tan KL, Yap SF

J Trop Pediatr, 2005 Dec;51(6):328-33.
PMID: 15967770 DOI: 10.1093/tropej/fmi052

beta-thalassaemia major, an autosomal recessive hemoglobinopathy, is one of the most common single gene disorders in multi-racial Malaysia. The control of beta-thalassaemia major requires a multi-disciplinary approach that includes population screening, genetic counselling, prenatal diagnosis and the option of termination of affected pregnancies. To achieve this objective, the molecular characterisation of the spectrum of beta-globin gene mutations in each of the affected ethnic groups is required. We studied 88 consecutive unrelated individuals and their respective families with beta-thalassaemia (74 beta-thalassaemia major, 12 HbE-beta-thalassaemia, 2 with HbE homozygotes) and four individuals with beta-thalassaemia trait that contributed a total 180 alleles for study. Using a 2-step molecular diagnostic strategy consisting of amplification refractory mutation system (ARMS) to identify the 8 most common mutations followed by other DNA-based diagnostic techniques, a total of 177 (98.3 per cent) of the 180 beta-thalassaemia alleles were characterised. One out of 91 (1 per cent) of the Chinese alleles, one out of 46 (2.2 per cent) Malay alleles and one out of two Indian alleles remained unknown. A 100 per cent success rate was achieved in studying the Kadazandusun community in this study. A strategy to identify beta-globin gene mutations in Malaysians with beta-thalassaemia is proposed based on this outcome.

Matched MeSH terms: DNA/genetics
Molecular characterization of a polymorphic 3-Mb deletion at chromosome Yp11.2 containing the AMELY locus in Singapore and Malaysia populations

Yong RY, Gan LS, Chang YM, Yap EP

Hum Genet, 2007 Nov;122(3-4):237-49.
PMID: 17588179

Amelogenin paralogs on Chromosome X (AMELX) and Y (AMELY) are commonly used sexing markers. Interstitial deletion of Yp involving the AMELY locus has previously been reported. The combined frequency of the AMELY null allele in Singapore and Malaysia populations is 2.7%, 0.6% in Indian and Malay ethnic groups respectively. It is absent among 541 Chinese screened. The null allele in this study belongs to 3 Y haplogroups; J2e1 (85.7%), F* (9.5%) and D* (4.8%). Low and high-resolution STS mapping, followed by sequence analysis of breakpoint junction confirmed a large deletion of 3 to 3.7-Mb located at the Yp11.2 region. Both breakpoints were located in TSPY repeat arrays, suggesting a non-allelic homologous recombination (NAHR) mechanism of deletion. All regional null samples shared identical breakpoint sequences according to their haplogroup affiliation, providing molecular evidence of a common ancestry origin for each haplogroup, and at least 3 independent deletion events recurred in history. The estimated ages based on Y-SNP and STR analysis were approximately 13.5 +/- 3.1 kyears and approximately 0.9 +/- 0.9 kyears for the J2e1 and F* mutations, respectively. A novel polymorphism G > A at Y-GATA-H4 locus in complete linkage disequilibrium with J2e1 null mutations is a more recent event. This work re-emphasizes the need to include other sexing markers for gender determination in certain regional populations. The frequency difference among global populations suggests it constitutes another structural variation locus of human chromosome Y. The breakpoint sequences provide further information to a better understanding of the NAHR mechanism and DNA rearrangements due to higher order genomic architecture.

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 A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library

Lim VC, Ramli R, Bhassu S, Wilson JJ

PLoS One, 2017;12(7):e0179555.
PMID: 28742835 DOI: 10.1371/journal.pone.0179555

Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.

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
Colorimetric detection of DNA hybridization based on a dual platform of gold nanoparticles and graphene oxide

Thavanathan J, Huang NM, Thong KL

Biosens Bioelectron, 2014 May 15;55:91-8.
PMID: 24368225 DOI: 10.1016/j.bios.2013.11.072

The unique property of gold nanoparticles (Au NP) to induce colour change and the versatility of graphene oxides (GO) in surface modification makes them ideal in the application of colorimetric biosensor. Thus we developed a label free optical method to detect DNA hybridization through a visually observed colour change. The Au NP is conjugated to a DNA probe and is allowed to hybridize with the DNA target to the GO thus causing a change in colour from pinkish-red to purplish blue. Spectrophometry analysis gave a wavelength shift of 22 nm with 1 µM of DNA target. Sensitivity testing using serially diluted DNA conjugated GO showed that the optimum detection was at 63 nM of DNA target with the limit at 8 nM. This proves the possibility for the detection of DNA hybridization through the use of dual nanoparticle system by visual observation.

Matched MeSH terms: DNA/genetics*
Fulltext Finding the factors of reduced genetic diversity on X chromosomes of Macaca fascicularis: male-driven evolution, demography, and natural selection

Osada N, Nakagome S, Mano S, Kameoka Y, Takahashi I, Terao K

Genetics, 2013 Nov;195(3):1027-35.
PMID: 24026095 DOI: 10.1534/genetics.113.156703

The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian-Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.

Matched MeSH terms: DNA/genetics
The genetic structure of Oreochromis spp. (Tilapia) populations in Malaysia as revealed by microsatellite DNA analysis

Bhassu S, Yusoff K, Panandam JM, Embong WK, Oyyan S, Tan SG

Biochem Genet, 2004 Aug;42(7-8):217-29.
PMID: 15487586

The genetic make-up of five populations of Oreochromis spp. was examined by microsatellite analysis. Eleven polymorphic microsatellite loci showed significant departures from the Hardy-Weinberg equilibrium. The mean heterozygosity ranged from 0.6280 to 0.7040 for each population. The genetic distance values showed a clear separation between O. niloticus and O. mossambicus. The differentiation of the O. niloticus populations was then tested with various genetic measures, which are based on both the Infinite Allele and the Stepwise Mutation models. All these measures grouped the populations similarly.

Matched MeSH terms: DNA/genetics
Heterogeneity in alpha-thalassemia interactions in Malays, Chinese and Indians in Malaysia

Wee YC, Tan KL, Chow TW, Yap SF, Tan JA

J Obstet Gynaecol Res, 2005 Dec;31(6):540-6.
PMID: 16343256 DOI: 10.1111/j.1447-0756.2005.00333.x

AIM: Interactions between different determinants of alpha-thalassemia raises considerable problems, particularly during pregnancies where antenatal diagnosis is necessary. This study aims to determine the different types of deletional alpha-thalassemia and Hemoglobin Constant Spring (HbCS), and their frequency in Malays, Chinese and Indians in Malaysia.
METHODS: DNA from 650 pregnant women from the Antenatal Clinic of the University of Malaya Medical Center in Kuala Lumpur, Malaysia who showed mean cell volume < or =89 fL and/or mean cell hemoglobin < or =28 pg were analyzed for the double alpha-globin gene South-East Asian deletion (--SEA), the -alpha3.7 and -alpha4.2 single alpha-globin gene deletions and HbCS.
RESULTS: One hundred and three (15.8%) of the pregnant women were confirmed as alpha-thalassemia carriers: 25 (3.8%) were alpha-thalassemia-1 carriers with the --SEA/alphaalpha genotype, 64 (9.8%) were heterozygous for the -alpha3.7 rightward deletion (-alpha3.7/alphaalpha), four (0.6%) were heterozygous for the -alpha4.2 leftward deletion (-alpha4.2/alphaalpha), nine (1.4%) were heterozygous for HbCS (alphaCSalpha/alphaalpha) and one (0.2%) was compound heterozygous with the -alpha3.7/alphaCSalpha genotype. The double alpha-globin gene --SEA deletion was significantly higher in the Chinese (15%) compared to the Malays (2.5%) and not detected in the Indians studied. The -alpha3.7 deletion was distributed equally in the three races. HbCS and -alpha4.2 was observed only in the Malays.
CONCLUSION: The data obtained gives a better understanding of the interactions of the different alpha-thalassemia determinants in the different ethnic groups, thus enabling more rapid and specific confirmation of alpha-thalassemia in affected pregnancies where antenatal diagnosis is necessary.
Study site: Antenatal clinic, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia

Matched MeSH terms: DNA/genetics
Fulltext High throughput molecular confirmation of β-thalassemia mutations using novel TaqMan probes

Kho SL, Chua KH, George E, Tan JA

Sensors (Basel), 2013;13(2):2506-14.
PMID: 23429513 DOI: 10.3390/s130202506

β-Thalassemia is a public health problem where 4.5% of Malaysians are β-thalassemia carriers. The genetic disorder is caused by defects in the β-globin gene complex which lead to reduced or complete absence of β-globin chain synthesis. Five TaqMan genotyping assays were designed and developed to detect the common β-thalassemia mutations in Malaysian Malays. The assays were evaluated with 219 "blinded" DNA samples and the results showed 100% sensitivity and specificity. The in-house designed TaqMan genotyping assays were found to be cost- and time-effective for characterization of β-thalassemia mutations in the Malaysian population.

Matched MeSH terms: DNA/genetics
Non-Invasive DNA Sampling for Molecular Analysis of Beta-Thalassemia: Amiable Alternative Sampling Methods with Accurate Results for Pediatric Patients

Abd Rahim MR, Kho SL, Kuppusamy UR, Tan JA

Clin. Lab., 2015;61(9):1325-30.
PMID: 26554253

BACKGROUND: Beta-thalassemia is the most common genetic disorder in Malaysia. Confirmation of the β-globin gene mutations involved in thalassemia is usually carried out by molecular analysis of DNA extracted from leukocytes in whole blood. Molecular analysis is generally carried out when affected children are around 1 - 2 years as clinical symptoms are expressed during this period. Blood taking at this age can be distressing for the child. High yield and pure DNA extracted from non-invasive sampling methods can serve as alternative samples in molecular studies for genetic diseases especially in pediatric cases.
METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.

Matched MeSH terms: DNA/genetics

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