The D1S80 allele frequencies in 124 unrelated Malays from the Malaysian population were determined and 51 genotypes and 19 alleles were encountered. The D1S80 frequency distribution met Hardy-Weinberg expectations. The observed heterozygosity was 0.80 and the power of discrimination was 0.96.
The allele frequencies for the apolipoprotein B (apo B) 5'-Ins/Del and 3'-VNTR polymorphisms varied significantly (p < 0.01) among Singaporeans of Chinese, Malay and Indian descent. We calculated the unbiased expected heterozygosities for the 5'-Ins/Del polymorphism as 0.3357, 0.1984 and 0.2418, and for the 3'-VNTR as 0.5980, 0.5260 and 0.6749, respectively, in the Chinese, Malays and Indians. Compared to heterozygosities reported for other populations, the Singaporeans differed from most Caucasians in having significantly lower values but were closely related to other non-Caucasians. Thirteen alleles, with a bimodal distribution, were observed at the 3'-VNTR polymorphic locus; the alleles occurring most frequently among the Chinese and Malays were of 35 or 53 repeats, and among the Indians, of 37 or 47 repeats. The Del allele was associated with elevated serum cholesterol (p = 0.023), LDL-cholesterol (LDL-C) (p = 0.001) in the Chinese, and apo B (p = 0.007) in the Indians. Likewise, the larger 3'-VNTR alleles (> 41 repeats) were associated with raised cholesterol (p = 0.018), LDL-C (p = 0.025), and triglyceride (p = 0.001) in the Chinese. The two polymorphisms were not in significant linkage disequilibrium (D = -0.0029, p = 0.494) in the three ethnic groups.
Allele frequencies for the nine STRs genetic loci included in the AmpFlSTR Profiler kit were obtained from samples of unrelated individuals comprising 139-156 Malays, 149-153 Chinese and 132-135 Indians, residing in Malaysia.
A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.
Allogeneic stem cell transplantation is a treatment option for malignant and non-malignant disorders in children. For children with no HLA-matched sibling or related stem cell donors, there is the option of unrelated cord blood donors. At the University of Malaya Medical Centre (UMMC) in Kuala Lumpur, the first unrelated cord blood transplantation (CBT) was performed in October 1997. All unrelated CBT performed in UMMC relied on cord blood units imported from overseas. DNA typing with variable number of tandem repeat (VNTR) loci was done to qualitatively evaluate engraftment in 15 unrelated CBT. In all the fifteen cases that were evaluated, molecular evidence of engraftment or non-engraftment correlated with the clinical findings.
Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.
The molecular genetic marker, minisatellite locus D1S80 (1p35-p36), is a highly polymorphic variable number of tandem repeats (VNTR). Its polymorphic nature allows for phylogenetic studies, forensic analysis, genetic maps construction and paternity testing to be performed. A study of the hypervariable locus D1S80 was conducted to determine the allele frequency and distribution of this locus in modern Malay in Sarawak population. The polymerase chain reaction technique was employed and results were analysed on polyacrylamide gel. A total of seventy-six DNA samples of unrelated Malay individuals in UNIMAS were collected and examined. The VNTR analysis of the D1S80 locus demonstrated the presence of 17 alleles in the Malay population. Allele with the size of 577 bp (27 repeats) was determined to be the most common in the sample population with the frequency of 0.1641, followed by allele with the size of 561 bp (26 repeats) and 529 bp (24 repeats) whose frequency is 0.1172 and 0.1094, respectively. The smallest allele is allele with the size of 465 bp (20 repeats) whereas the largest is allele with the size of 753 bp (38 repeats). The sample population exhibited 57.8% heterozygosity.
Following hematopoietic stem cell transplantation (HSCT), it is important to determine whether engraftment is successful and to track the dynamic changes of the graft. Tandem repeats such as minisatellites and microsatellites are currently the most established markers for chimerism application. We have developed a reliable method to quantitatively evaluate engraftment status in post-allogeneic HSCT patients using variable number of tandem repeat (VNTR) markers and "lab-on-a-chip" microfluidic electrophoresis technology. Following identification of an informative marker by conventional polymerase chain reaction (PCR), donor chimerism percentage was calculated based on a standard curve generated from artificially mixed patient-donor DNA-specific alleles in serial dilutions. All PCR products were mixed with commercial gel dye and loaded into Agilent DNA 1000 microfluidic LabChips for DNA sizing and quantitation. In 44 patients, separation of pretransplant and donor DNA fragments was resolved clearly and accomplished rapidly within 30min. Chimerism analysis using this platform is able to detect an amount as low as 6.3% donor DNA with acceptable coefficient of variation values. We also demonstrated concordant chimerism analysis findings using both microchip tandem repeats and real-time PCR quantitation of insertion-deletion polymorphisms. This microchip platform obviates the need for fluorescently labeled primers or any post-PCR sample manipulation. Quantitative monitoring of post-HSCT chimerism status using microfluidic electrophoresis is a useful tool for both large- and small-scale post-HSCT chimerism centers.
Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F = 0.63) while PFGE generated 35 pulsotypes (F = 0.71). Simpson's index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D = 0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P < .01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation.
A total of 78 alleles and 29 loci were detected from nine microsatellite and three minisatellite markers, respectively across 26 blast and ufra disease resistant genotypes. For blast resistant genotypes, the Polymorphic Information Content (PIC) values ranged from 0.280 to 0.726 and RM21 was considered as the best marker. PIC values ranged from 0.5953 to 0.8296 for ufra resistant genotypes and RM23 was the best marker for characterization of ufra resistant genotypes. The genetic similarity analysis using UPGMA clustering generated nine clusters with coefficient of 0.66 for blast resistant genotypes while five genetic clusters with similarity coefficient of 0.42 for ufra resistant genotypes. In order to develop resistant varieties of two major diseases of rice, hybridisation should be made using the parents, BR29 and NJ70507, BR36 and NJ70507 for blast, while BR11 and Aokazi, BR3 and Aokazi, Rayda and BR3 and Rayda and BR11 for ufra.
Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.
Vanda Mimi Palmer (VMP) is a highly sought as fragrant-orchid hybrid in Malaysia. It is economically important in cosmetic and beauty industries and also a famous potted ornamental plant. To date, no work on fragrance-related genes of vandaceous orchids has been reported from other research groups although the analysis of floral fragrance or volatiles have been extensively studied. An expressed sequence tag (EST) resource was developed for VMP principally to mine any potential fragrance-related expressed sequence tag-simple sequence repeat (EST-SSR) for future development as markers in the identification of fragrant vandaceous orchids endemic to Malaysia. Clustering, annotation and assembling of the ESTs identified 1,196 unigenes which defined 966 singletons and 230 contigs. The VMP dbEST was functionally classified by gene ontology (GO) into three groups: molecular functions (51.2%), cellular components (16.4%) and biological processes (24.6%) while the remaining 7.8% showed no hits with GO identifier. A total of 112 EST-SSR (9.4%) was mined on which at least five units of di-, tri-, tetra-, penta-, or hexa-nucleotide repeats were predicted. The di-nucleotide motif repeats appeared to be the most frequent repeats among the detected SSRs with the AT/TA types as the most abundant among the dimerics, while AAG/TTC, AGA/TCT-type were the most frequent trimerics. The mined EST-SSR is believed to be useful in the development of EST-SSR markers that is applicable in the screening and characterization of fragrance-related transcripts in closely related species.
This study describes the organization of the repetitive pattern in the mtDNA control region of Tomistoma schlegelii. Using newly designed primers, we detected length variations of approximately 50-100 bp among individuals, and only one individual showed a heteroplasmic band. Sequencing the region after CSB III revealed two main patterns: a repeat motif and a variable number tandem repeat (VNTR) pattern. The VNTR region, with a core unit of 104 bp, consisting of four motifs and a short AT chain, is implicated in the length variation seen among individuals of Tomistoma. A conserved motif seen in a family unit indicated that the repeat pattern was stably inherited from the maternal parent to all offspring. A combination of VNTR patterns specific to different crocodilians was seen in Tomistoma, and the overall secondary structure was shown to be similar to that in Crocodylus and Gavialis.
Variable number of tandem repeats (VNTR) polymorphism in the interleukin 4 (IL-4) gene has been associated with end-stage renal disease (ESRD) subjects in many different populations, although with conflicting results. We determined the 70 bp of VNTR polymorphism at intron 3 of the IL-4 gene in Malaysian ESRD subjects. Buccal cells were collected from 160 case and 160 control subjects; genomic DNA was amplified using PCR, followed by agarose gel electrophoresis. There were significant differences in genotypes and alleles of the IL-4 gene. We conclude that VNTR polymorphism of the IL-4 gene is a risk factor for the development of ESRD among Malaysians.
Human endothelial nitric oxide synthase (eNOS) is one isoform of the nitric oxide synthases that are responsible for nitric oxide synthesis from L-arginine. The gene encoding eNOS contains a 27-bp VNTR polymorphism in intron 4. We report here for the first time the presence of a novel allele 3, which was absent in all other populations studied to date, in 1.7% each of Singaporean Indians and Malays. We also detected the presence of a novel genotype 3/5 in 3.4% each of Singaporean Indians and Malays. Allele 6, which was absent in Han Chinese from northern China and Taiwan and was also absent in Indians from the Indian subcontinent, was found in 2.1% of Singaporean Chinese and in 0.3% of Singaporean Indians.
A variable-number tandem-repeat (VNTR) typing assay for the differentiation of Mycobacterium abscessus strains was developed. This assay showed complete reproducibility, locus stability, and a discriminatory power (Hunter-Gaston discriminatory index [HGDI] of 0.9563) that is superior to that of multilocus sequencing. It is a promising tool for the investigation of Mycobacterium abscessus epidemiology and nosocomial outbreaks.
Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common causative agent of non-typhoidal salmonellosis in Malaysia. We aimed to characterize S. Enteritidis isolated from humans and animals by analyzing their antimicrobial resistance profiles and genotypes. A total of 111 strains were characterized using multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and antimicrobial susceptibility testing. Both typing methods revealed that genetically similar S. Enteritidis strains had persisted among human and animal populations within the period of study (2003-2008). Only 39% of the strains were multi-drug resistant (i.e., resistant to 3 or more classes of antimicrobial agents), with a majority (73%) of these in low-risk phase (multiple antibiotic resistant index <0.20). Limited genetic diversity among clinical and zoonotic S. Enteritidis suggested that animals are possible sources of human salmonellosis. The degree of multi-drug resistance among the strains was generally low during the study period.
Mycobacterium abscessus is a rapidly growing mycobacterium that is often associated with human infections. The taxonomy of this species has undergone several revisions and is still being debated. In this study, we sequenced the genomes of 12 M. abscessus strains and used phylogenomic analysis to perform subspecies classification.
Salmonella Typhimurium is an important nontyphoidal Salmonella serovar associated with foodborne diseases in many parts of the world. This organism is the major causative agent of nontyphoidal salmonellosis in Malaysia. We aimed to investigate the genetic profiles of the strains isolated from clinical, zoonotic, and dietary sources in Malaysia using multilocus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). By focusing on the 5 common variable number tandem repeat (VNTR) loci, we found that PFGE (D = 0.99) was more discriminative than MLVA (D = 0.76). The low MLVA score might be because of a lack of VNTR loci STTR6 (81.0%) and STTR10pl (76.2%). Both subtyping methods suggested that our S. Typhimurium strains were largely endemic with limited genetic variation. Furthermore, we observed that biphasic S. Typhimurium strains were dominant (99%) and multidrug resistance was prevalent (50%) within our sample pool. The most frequently observed phenotypes were resistance to compound sulfonamides (49%), tetracycline (51%), and streptomycin (52%). In this study, we documented the genetic relationship, antimicrobial resistance characteristics, and flagellar-phase dominance among S. Typhimurium strains found in Malaysia.
The aim of this study was to determine the association of the c.894G>T; p.Glu298Asp polymorphism and the variable number tandem repeat (VNTR) polymorphism of the endothelial nitric oxide synthase (eNOS) gene and c.181C>T polymorphism of the bradykinin type 2 receptor gene (B2R) in Malaysian end-stage renal disease (ESRD) subjects. A total of 150 ESRD patients were recruited from the National Kidney Foundation's (NKF)dialysis centers in Malaysia and compared with 150 normal healthy individuals. Genomic DNA was extracted from buccal cells of all the subjects. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was carried out to amplify the products and the restricted fragments were separated by agarose gel electrophoresis. Statistical analyses were carried out using software where a level of p <0.05 was considered to be statistically significant. The genotypic and allelic frequencies of the B2R gene (c.181C>T, 4b/a) and eNOS gene (c.894G>T) polymorphisms were not statistically significant (p >0.05) when compared to the control subjects. The B2R and eNOS gene polymorphisms may not be considered as genetic susceptibility markers for Malaysian ESRD subjects.