Copy number variation (CNV) is a form of genetic variation in addition to single nucleotide polymorphisms. The significance of CNV in the manifestation of a number of diseases is only recently receiving considerable attention. We genotyped 163 dengue patients from Peninsular Malaysia for genes possibly linked to dengue infection using quantitative real-time PCR. Here, we report a serendipitous discovery of a novel rare CNV of the ABCF1 gene among the dengue patients. Among these patients, two had a gain of 1 copy (CN = 3) and one had lost 1 copy (CN = 1), indicating that a rare CNV of the ABCF1 gene was detected among dengue patients from Peninsular Malaysia. Although the gene is suspected to regulate inflammatory responses and pathogen-induced cytokine storm, its relevance to dengue requires further investigation.
To examine the impact of regional and seasonal variations on the incidence and severity of sheath rot, a major seed-borne disease of rice caused by Sarocladium oryzae, data on incidence and severity were collected from 27 selected fields in the Gazipur, Rangpur, Bogra, Chittagong, Comilla, Gopalgonj, Jessore, Manikgonj, and Bhola districts of Bangladesh in rain-fed and irrigated conditions. Cultural variability of 29 pathogen isolates obtained from 8 different locations was studied on potato dextrose agar (PDA) and genetic variability was determined by DNA fingerprinting using variable number tandem repeat-polymerase chain reaction markers. Overall, disease incidence and severity were higher in irrigated rice. Disease incidence and severity were highest in the Bhola district in rain-fed rice and lowest in irrigated rice. Mycelial growth of 29 representative isolates was found to vary on PDA and the isolates were divided into 6 groups. The range of the overall size of conidia of the selected isolates was 2.40-7.20 x 1.20-2.40 μm. Analysis of the DNA fingerprint types of the 29 isolates of S. oryzae, obtained from the amplification reactions, revealed 10 fingerprinting types (FPTs) that were 80% similar. FPT-1 was the largest group and included 13 isolates (44.8%), while FPT-2 was the third largest group and included 3 isolates. Each of FPT-3, 4, 5, and 6 included only 1 isolate. We observed no relationship between cultural and genetic groupings.
We developed an alternative method to extract DNA and RNA from clotted blood for genomic and molecular investigations. A combination of the TRIzol method and the QIAamp spin column were used to extract RNA from frozen clotted blood. Clotted blood was sonicated and then the QIAamp DNA Blood Mini Kit was used for DNA extraction. Extracted DNA and RNA were adequate for gene expression analysis and copy number variation (CNV) genotyping, respectively. The purity of the extracted RNA and DNA was in the range of 1.8-2.0, determined by absorbance ratios of A(260):A(280). Good DNA and RNA integrity were confirmed using gel electrophoresis and automated electrophoresis. The extracted DNA was suitable for qPCR and microarrays for CNV genotyping, while the extracted RNA was adequate for gene analysis using RT-qPCR.
Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.
The identification of new biomarkers for early detection of highly recurrent head and neck cancer is urgently needed. MicroRNAs (miRNAs) are small and non-coding RNAs that regulate cancer-related gene expression, such as tumor protein 53 (TP53) gene expression. This study was carried out to analyze TP53 gene expression using real-time PCR and to determine changes in intracellular p53 level by flow cytometry after downregulation of miRNA-181a miRNA inhibitor in the FaDu cell line. TP53 gene expression showed a 3-fold increment and the p53 protein level was also increased in the miRNA-181a-treated cells. In conclusion, miRNA-181a binds to the TP53 gene and inhibits its expression, decreasing the synthesis of p53.
Genetic diversity and patterns of population structure of the 94 oil palm lines were investigated using species-specific simple sequence repeat (SSR) markers. We designed primers for 63 SSR loci based on their flanking sequences and conducted amplification in 94 oil palm DNA samples. The amplification result showed that a relatively high level of genetic diversity was observed between oil palm individuals according a set of 21 polymorphic microsatellite loci. The observed heterozygosity (Ho) was 0.3683 and 0.4035, with an average of 0.3859. The Ho value was a reliable determinant of the discriminatory power of the SSR primer combinations. The principal component analysis and unweighted pair-group method with arithmetic averaging cluster analysis showed the 94 oil palm lines were grouped into one cluster. These results demonstrated that the oil palm in Hainan Province of China and the germplasm introduced from Malaysia may be from the same source. The SSR protocol was effective and reliable for assessing the genetic diversity of oil palm. Knowledge of the genetic diversity and population structure will be crucial for establishing appropriate management stocks for this species.
Type 2 diabetes mellitus (T2DM) is believed to be associated with excessive production of reactive oxygen species. Glutathione S-transferase (GST) polymorphisms result in decreased or absent enzyme activity and altered oxidative stress, and have been associated with cardiovascular disease (CVD). The present study assessed the effect of GST polymorphisms on the risk of developing T2DM in individuals of Malaysian Malay ethnicity. A total of 287 subjects, consisting of 87 T2DM and 64 CVD/T2DM patients, as well as 136 healthy gender- and age-matched controls were genotyped for selected polymorphisms to evaluate associations with T2DM susceptibility. Genomic DNA was extracted using commercially available kits, and GSTM1, GSTT1, and α-globin sequences were amplified by multiplex polymerase chain reaction. Biochemical parameters were measured with a Hitachi autoanalyzer. The Fisher exact test, the chi-square statistic, and means ± standard deviations were calculated using the SPSS software. Overall, we observed no significant differences regarding genotype and allele frequencies between each group (P = 0.224 and 0.199, respectively). However, in the combined analysis of genotypes and blood measurements, fasting plasma glucose, HbA1c, and triglyceride levels, followed by age, body mass index, waist-hip ratio, systolic blood pressure, and history of T2DM significantly differed according to GST polymorphism (P ˂ 0.05). Genetically induced absence of the GSTT1 enzyme is an independent and powerful predictor of premature vascular morbidity and death in individuals with T2DM, and might be triggered by cigarette smoking's oxidative effects. These polymorphisms could be screened in other ethnicities within Malaysia to determine further possible risk factors.
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.
Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position.
Systemic lupus erythematosus (SLE) is an autoimmune disease that causes systemic damage, involving auto-reactive antibodies and over-deposition of immune complexes. Susceptibility to SLE is believed to be multifactorial, and genetics is one of the proven etiological factors; it can affect SLE development, severity and prognosis. We investigated a possible association between the angiotensin-converting enzyme gene and susceptibility to SLE in the Malaysian population. PCR was employed for the determination of I/D dimorphism of this gene. The I allele was more frequent than the D allele in both the SLE patients (N = 170) and healthy controls (N = 190). However, there was no significant difference in the distribution of these two alleles between both groups studied (χ(2) = 0.284, P > 0.05). Interestingly, the DD homozygous genotype scored notably higher in the healthy control group (χ(2) = 7.568, P < 0.05), while the ID heterozygote was observed to be significantly associated with SLE (χ(2) = 11.143, P < 0.05). In conclusion, with respect to the Malaysian population, the DD genotype might play a protective role in the development of SLE while in contrast, those who carry the ID genotype might be at potential risk for onset of this disease.
The genus Curcuma is a member of the ginger family (Zingiberaceae) that has recently become popular for use as flowering pot plants, both indoors and as patio and landscape plants. We used PCR-based molecular markers (SSRs) to elucidate genetic variation and relationships between five varieties of Curcuma (Curcuma alismatifolia) cultivated in Malaysia. Of the primers tested, 8 (of 17) SSR primers were selected for their reproducibility and high rates of polymorphism. The number of presumed alleles revealed by the SSR analysis ranged from two to six alleles, with a mean value of 3.25 alleles per locus. The values of HO and HE ranged from 0 to 0.8 (mean value of 0.2) and 0.1837 to 0.7755 (mean value of 0.5102), respectively. Eight SSR primers yielded 26 total amplified fragments and revealed high rates of polymorphism among the varieties studied. The polymorphic information content varied from 0.26 to 0.73. Dice's similarity coefficient was calculated for all pairwise comparisons and used to construct an unweighted pair group method with arithmetic average (UPGMA) dendrogram. Similarity coefficient values from 0.2105 to 0.6667 (with an average of 0.4386) were found among the five varieties examined. A cluster analysis of data using a UPGMA algorithm divided the five varieties/hybrids into 2 groups.
Growth factors are polypeptides that are critical for the initiation, progression, and metastasis of cancer. Most tumor cells are capable of synthesizing particular growth factors leading to constitutive pathway activation in these cells through autocrine signaling. Epidermal growth factor (EGF) is a potent mitogenic peptide that exerts direct effects on the proliferation and differentiation of tumor cells in carcinogenesis. By contrast, vascular endothelial growth factor (VEGF) is vital for the invasion and metastasis of neoplasms through the formation of new blood vessels from mature endothelial cells. In this study, we investigated the association between functional polymorphisms of both the EGF and VEGF genes and colorectal cancer (CRC) susceptibility. A total of 130 CRC patients and 212 healthy controls were recruited for this case-control study. Genotyping of genetic variants was conducted via real-time polymerase chain reaction (PCR) amplification with allele-specific TaqMan probes. None of the genotypes of the EGF +61 A>G and VEGF +936 C>T variants was significantly associated with CRC susceptibility among the Malaysian subjects evaluated (P > 0.05). The observed frequency distributions of the EGF +61 A>G polymorphism genotypes showed ethnic heterogeneity, which was not the case for the VEGF +936 C>T genotypes. In conclusion, no positive correlation between these functional polymorphisms and CRC risk was found in this Malaysian population. Studies of the EGF and VEGF genes and CRC susceptibility are scarce, and the results reported thus far differ from one population to another. Hence, more replication studies are warranted before any firm conclusions can be made.
Gastrointestinal cancers are malignant diseases with high mortality rate. Early diagnosis of patients could improve the results of treatment. Many studies used dermatoglyphics as a biomarker to predict the incidence of genetic diseases and cancers. This study assessed the association between gastrointestinal cancers and particular fingerprint patterns, which could be useful in early diagnosis of these malignancies. The study was conducted on 153 histopathologically confirmed gastrointestinal cancer patients and 299 healthy individuals. The fingerprints were taken by a specific method of rolling the subject's fingers or thumbs in ink. The data were analyzed for the significance using the chi-square test and the t-test. Odds ratio with 95% confidence intervals were calculated. Dermatoglyphic analysis showed that whorl and loop patterns significantly changed in the case group as compared to control. However, the odds ratio suggested that whorl pattern in 6 or more fingers might be a risk factor for developing gastrointestinal cancers. Our results showed that there is an association between fingerprint patterns and gastrointestinal cancers, and so, the dermatoglyphic analysis may aid in the early diagnosis of these cancers.
The aim of this study was to determine whether C34T, a common polymorphism of the adenosine monophosphate deaminase 1 gene (AMPD1), is associated with essential hypertension (EH). We hypothesize that C34T is associated with the development of EH. A case-control design was used for this study. The DNA was extracted using a commercial kit from the whole blood of 200 patients with hypertension and 200 subjects without hypertension from selected Malaysian ethnicities (Malays, Chinese, and Indians). Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) and agarose gel electrophoresis were used for genotyping. The C34T gene polymorphism of AMPD1 was significantly associated with EH in the Malaysian subjects (P < 0.0001). The genotype frequencies of CC, CT, and TT were 6%, 79%, and 15%, respectively, among hypertensive subjects, while no TT genotypes were observed in the normotensive subjects. Further, the frequency of hypertension was higher among T allele carriers than C carriers (OD = 9.94; 95%CI = 6.851-14.434). There were significant differences in the systolic blood pressure, diastolic blood pressure, and pulse pressure (P ˂ 0.05) between the normotensive and hypertensive Malaysian subjects; we believe those difference were caused by the C34T polymorphism. For the first time in Malaysia, the current study provides evidence that a common polymorphism of the AMPD1 gene (C34T) is strongly associated with EH.
We evaluated the possible influence of glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genes on genetic damage due to occupational exposure, which contributes to accelerate ageing. This study was conducted on 120 car auto repair workshop workers exposed to occupational hazards and 120 controls without this kind of exposure. The null and non-null genotypes of GSTM1 and GSTT1 genes were determined by multiplex PCR. Micronucleus frequency, Comet tail length and relative telomere length differences between the null and non-null genotypes of the GSTM1 gene were significantly greater in the exposed group. Lack of GSTT1 did not affect the damage biomarkers significantly (P > 0.05), while lack of GSTM1 was associated with greater susceptibility to genomic damage due to occupational exposure. It was concluded that early ageing is under the influence of these genes and the environmental and socio-demographic factors. Duration of working time was significantly associated with micronucleus frequency, Comet tail length and relative telomere length.
This study aims to investigate the effects of tumor necrosis factor alpha (TNF-α) G308A gene polymorphism on essential hypertension (EHT) with or without type 2 diabetes mellitus (T2DM). The project was conducted on buccal epithelial and blood cells for case and control patients, respectively. Epithelial cells were obtained from the inner part of the cheeks. Techniques including DNA extraction, polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLP) were utilized to assess biomarkers of DNA damage. Our results demonstrated significant differences between wild and mutated genotypes among EHT patients without T2DM. We also found a significant association between wild and mutated allele frequencies in EHT patients (P < 0.05). Clinical characteristics between the groups (EHT with or without T2DM and controls) showed statistically significant association (P < 0.05). Overall, we show that G308A polymorphism of the TNF-αgene may be a significant genetic risk factor for EHT without T2DM patients in Malaysia.
The sympathetic nervous system plays a major role in blood pressure regulation. Beta 2 (β2) adrenoceptor gene polymorphisms have been associated with hypertension in different populations with conflicting results. We examined the association of three common polymorphisms, Arg16Gly, Gln27Glu, and Thr164Ile, of the β2 adrenoceptor gene in Malaysian hypertensive subjects. A total of 160 hypertensive and control subjects were recruited. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and anthropometric measurements were obtained from each subject. Biochemical analyses of lipid profiles were conducted with an autoanalyzer. DNA samples were extracted from blood and buccal cells. Genotyping was accomplished with polymerase chain reaction-restriction fragment length polymorphism. SBP, DBP, body mass index, and biochemical factors all differed significantly between case and control subjects (P < 0.05). The genotype frequencies of Arg16Arg, Arg16Gly, and Gly16Gly were 22.5, 70, and 7.5% among cases and 33.1, 63.1, and 3.8% among controls, respectively. The genotype frequencies of Gln27Gln, Gln27Glu, and Glu27Glu among cases were 41.1, 50, and 1.9% compared to 77.5, 20.6, and 1.9% among controls, respectively. In this study, the Gln27Glu polymorphism was significantly associated with Malaysian hypertensive subjects (P < 0.05). Therefore, the Gln27Glu polymorphism of the β2 adrenoceptor could be a risk factor associated with hypertension among Malaysians.
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.
A number of studies have pointed to the association of BDNF (brain-derived neurotrophic factor) and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa) with schizophrenia. The purpose of this study was to determine whether these two genes are involved in the pathogenesis of schizophrenia in the Malay population. Two single nucleotide polymorphisms Val66Met of BDNF, -2036C>G and g.1238delG of DARPP-32 were genotyped in the Malay population in 200 patients with schizophrenia and 256 healthy controls. Analysis of allele and genotype frequencies in these two groups revealed no significant association of BDNF or DARPP-32 polymorphisms with schizophrenia in Malays. This is the first such association study in the Malay population.
This study was carried out to evaluate the genetic effect of quantitative trait loci (QTLs) conferring drought tolerance in wheat. A population of 120 F(2) individuals from the cross between the drought-tolerant S-78-11 and drought-sensitive Tajan cultivars were analyzed for their segregation under drought stress conditions. The relative water content under drought stress conditions exhibited continuous variation, indicating the minor gene effects on the trait. Single-marker analysis (SMA) was carried out to detect the main QTL association with drought tolerance. The SMA results revealed that the simple sequence repeat markers GWM182 and GWM292 on chromosome 5D and GWM410 on chromosome 5A exhibited significant association with drought tolerance, accounting for 30, 22, and 21% of the total variation, respectively. The 3 genetic loci, especially GWM182, can be used in marker-assisted selection methods in drought tolerance breeding in wheat.