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The association of mitochondrial DNA 5178 C > a polymorphism with plasma lipid levels among three ethnic groups

Lal S, Madhavan M, Heng CK

Ann. Hum. Genet., 2005 Nov;69(Pt 6):639-44.
PMID: 16266403

Mitochondria are eukaryotic cytoplasmic organelles responsible for oxidative phosphorylation. The C to A nucleotide transversion in the NADH dehydrogenase subunit 2 (MT-ND2) coding region of mitochondrial DNA has been reported to be associated with plasma lipid levels, adult onset diseases and longevity. We have examined the role of this polymorphism in relation to plasma lipid levels and age in a total of 713 healthy individuals belonging to 3 ethnic groups in Singapore. The frequency of the A allele was significantly higher (p < 0.05) among the Chinese (0.15) in comparison to the Malays (0.05) and Indians (0.02). No significant difference in the frequency of the allele was observed between healthy and coronary artery disease subjects, and between age-stratified subjects. We found that the polymorphism is significantly associated in an ethnic- and gender-specific manner with plasma apoB levels in the Chinese males (p < 0.05). This is the first epidemiological report of the mt5178 C > A polymorphism and its association with plasma lipid levels in Asian populations outside Japan.
Bioinformatics in molecular immunology laboratories demonstrated: Modeling an anti-CMV scFv antibody

Heng CK, Othman RY

Bioinformation, 2006 Mar 27;1(4):118-20.
PMID: 17597869

A scFv (single chain variable fragment) antibody clone from anti-CMV (anti-cucumber mosaic virus) was successfully constructed from immunized mouse and the DNA sequence was submitted to GenBank (AY337618 and AY337619). The expression of a 32 kDa recombinant antibody in bacteria was verified using ELISA (enzyme-linked immunoassay) and western blot. However, elucidation of specific anti-CMV scFv function requires detailed and time consuming immuno-assays. Alternatively, useful functional information on anti-CMV scFV antibody can be obtained using available Bioinformatics tools and techniques without performing tedious assays. Here, we use the commonly used Bioinformatics tools and databases such as BLAST (basic local alignment search tool), GenBank, PDB (protein databank), KABAT numbering, SWISS-MODEL and Insight II to gain specific functional insights into anti-CMV scFv.
Fulltext Stacking gels: A method for maximising output for pulsed-field gel electrophoresis

Heng SK, Heng CK, Puthucheary SD

Indian J Med Microbiol, 2009 Apr-Jun;27(2):142-5.
PMID: 19384038 DOI: 10.4103/0255-0857.49428

Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.
The effects of three factor VII polymorphisms on factor VII coagulant levels in healthy Singaporean Chinese, Malay and Indian newborns

Quek SC, Low PS, Saha N, Heng CK

Ann. Hum. Genet., 2006 Nov;70(Pt 6):951-7.
PMID: 17044869

Factor VII (FVII) is an independent risk factor for coronary artery disease. Three polymorphisms of the factor VII gene (F7) were studied in a group of healthy newborns comprising 561 Chinese, 398 Malays and 226 Asian Indians from Singapore. The allele frequencies of 3 polymorphisms (R353Q, Promoter 0/10bp Del/Ins and Intron 7) in the FVII gene were ascertained through genotyping by polymerase chain reaction and restriction digestion of amplified fragments. In Chinese the minor allele frequencies are Q: 0.04, Ins: 0.03, R7: 0.44; Malays, Q: 0.06, Ins: 0.10, R7: 0.41; and Indians, Q: 0.25, Ins: 0.23, R7: 0.43. Strong linkage disequilibrium (Delta > 0.7) is observed between the 0/10 bp and the R353Q sites in all ethnic groups. We conclude that: (i) the prevalence of the minor Q and Ins alleles of the R353Q and 0/10 bp polymorphisms are significantly higher in the Indian newborns than the Chinese and Malays; (ii) the Q allele is significantly associated (p = 0.01) with a lower plasma FVII coagulant level in the Indian and Malay neonates; and this polymorphism explains up to 3.8% of the variance in FVII coagulant levels; (iii) there is no significant difference in allele frequencies of the three polymorphisms between neonates with and without family histories of CAD.
The Medicinal Mushroom Ganoderma neo-japonicum (Agaricomycetes) from Malaysia: Nutritional Composition and Potentiation of Insulin-Like Activity in 3T3-L1 Cells

Subramaniam S, Sabaratnam V, Heng CK, Kuppusamy UR

Int J Med Mushrooms, 2020;22(1):65-78.
PMID: 32463999 DOI: 10.1615/IntJMedMushrooms.2020033250

Ganoderma neo-japonicum is an annual polypore mushroom that is consumed by Malaysian indigenous tribes to treat various ailments including diabetes. The present study aimed to investigate the nutritive composition and in vitro antihyperglycemic effects of G. neo-japonicum extracts on 3T3-L1 preadipocytes. Nutritional analysis of G. neo-japonicum basidiocarps indicated a predominant presence of carbohydrates, proteins, dietary fiber, and microelements. Hot aqueous extract (AE) and its isolated (1,3)(1,6)-β-D-glucan polysaccharide (GNJP) from basidiocarps of G. neo-japonicum were evaluated for their ability to stimulate insulin independent adipogenesis, glucose uptake, adiponectin secretion, and regulate gene expression in 3T3-L1 adipocytes. GNJP showed a dose dependent stimulation of glucose uptake and adiponectin secretion but attenuated lipid accumulation in 3T3-L1 adipocytes. It upregulated the expressions of adiponectin, Aktl (protein kinase B), PPARγ (peroxisome proliferator activated receptor gamma), PRKAG2 (protein kinase, AMP activated), and Slc2a4 (glucose transporter) genes to stimulate glucose uptake in 3T3-L1 cells, which may have contributed to the insulin-mimicking activities observed in this study. In summary, the nutritive compositions and significant glucose uptake stimulatory activities of GNJP indicated that it may have potential use in the formulation of functional food for the management of hyperglycemia, insulin resistance, and related complications.
Functional Properties of Partially Characterized Polysaccharide from the Medicinal Mushroom Ganoderma neo-japonicum (Agaricomycetes)

Subramaniam S, Raman J, Sabaratnam V, Heng CK, Kuppusamy UR

Int J Med Mushrooms, 2017;19(10):849-859.
PMID: 29256840 DOI: 10.1615/IntJMedMushrooms.2017024355

This study was conducted to evaluate the mycochemical composition and antiglycemic and antioxidant activities of Ganoderma neo-japonicum hot aqueous extracts, prepared at different boiling durations, and polysaccharides isolated from them. Ground basidiocarps of G. neo-japonicum were double-boiled at 100°C for 0.5, 3, or 4 hours, and the antiglycemic activity was assessed by α-amylase and α-glucosidase enzyme inhibition assays. The antioxidant capacity of the crude hot aqueous extracts (AE-1, AE-2, AE-3) was assessed by DPPH and ABTS radical scavenging and ferric-reducing antioxidant power assays. The total phenolics, protein, and sugar in the crude extracts were also determined. The hot aqueous extract (AE-3) containing a significant amount of total sugar and having enhanced antiglycemic and antioxidant activities was selected for polysaccharide isolation. The isolated crude polysaccharide was separated and purified using diethylaminoethyl-cellulose-52 and Sepharose 6B column chromatography. Fourier transform infrared spectroscopy studies of the purified polysaccharide fraction (PF) showed the presence of typical bands corresponding to polysaccharides. The estimated β-glucan concentration in the PF was 39.26%. In general, the PF exhibited significantly lower antioxidant activity than AE-3. Nevertheless, its potency in inhibiting carbohydratehydrolyzing enzymes may have potential in the management of diabetes mellitus.
Genotype associations among seven apolipoprotein B polymorphisms in a population of Orang Asli of western Malaysia

Gajra B, Candlish JK, Heng CK, Mak JW, Saha N

Hum Biol, 1997 Oct;69(5):629-40.
PMID: 9299883

Associations among seven apolipoprotein B (APOB) gene polymorphisms [C-T promoter site; Leu-Ala-Leu signal peptide (SP) insertion/deletion; AG C,G site at codon 71; AG A1,D site at codon 591; XbaI site at codon 2488; AG H,I site at codon 3611; and AG T,Z site at codon 4154] were investigated in 195 members of an Orang Asli (aborigine) population from western Malaysia. Frequencies of the rare alleles for all these polymorphisms turned out to be low when compared with European but not Asian populations. The AG H,I site was not polymorphic. The highly polymorphic sites are in linkage disequilibrium among themselves, as shown by their delta values: SP 24,27 and AG C,G, 0.68; SP 24,27 and AG A1,D, 0.71; XbaI and AG C,G, 0.64; XbaI and AG A1,D, 0.57; SP 24,27 and XbaI, 0.48; and AG C,G and AG A1,D, 0.68. Ten unequivocal haplotypes on the basis of six sites (excluding the promoter polymorphism) were observed, and they represent 80% of the sample. The frequency of haplotype SP27,G,A1,X-,I,T, defined by the common homozygotes at all the sites for the APOB gene was 0.7, compared with 0.22 in Europeans. The ancestral haplotype SP27,G,D,X-,I,T was present at low frequency (0.01) in both the Orang Asli and Europeans. A cladogram constructed on the basis of haplotypes in the Orang Asli shows two different lines of evolution and that other haplotypes evolved by subsequent mutations on the ancestral haplotype.
Thiopurine methyltransferase polymorphisms in a multiracial asian population and children with acute lymphoblastic leukemia

Kham SK, Tan PL, Tay AH, Heng CK, Yeoh AE, Quah TC

J Pediatr Hematol Oncol, 2002 Jun-Jul;24(5):353-9.
PMID: 12142782

The purpose of this study was to determine the frequency of thiopurine methyltransferase (TPMT) polymorphisms in a multiracial Asian population and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Six hundred unrelated cord blood samples from 200 Chinese, Malay, and Indian healthy newborns were collected at the National University Hospital, Singapore; an additional 100 children with ALL were analyzed for five of the commonly reported TPMT variant alleles using polymerase chain reaction/restriction fragment length polymorphism and allele-specific polymerase chain reaction-based assays. In the cord blood study, the TPMT*3C variant was detected in all three ethnic groups; Chinese, Malays, and Indians had allele frequencies of 3%, 2.3%, and 0.8%, respectively. The TPMT*3A variant was found only among the Indians at a low allele frequency of 0.5%. The TPMT*6 variant was found in one Malay sample. Among the children with ALL, two white and one Chinese were heterozygous for the TPMT*3A variant and showed intermediate sensitivity to 6-mercaptopurine during maintenance therapy. Three Chinese patients and one Malay patient were heterozygous for the TPMT*3C variant. Mercaptopurine sensitivity could be validated in only one out of four TPMT*3C heterozygous patients. The overall allele frequency of the TPMT variants in this multiracial population was 2.5%. The TPMT*3C was the most common variant allele; TPMT*3A and TPMT*6 were rare. These results support the feasibility of performing TPMT genotyping in all children diagnosed with acute leukemia to minimize toxicity from thiopurine chemotherapy.
Variation of the platelet glycoprotein IIIa PI(A1/A2) allele frequencies in the three ethnic groups of Singapore

Lim J, Lal S, Ng KC, Ng KS, Saha N, Heng CK

Int J Cardiol, 2003 Aug;90(2-3):269-73.
PMID: 12957761

BACKGROUND: Polymorphisms of the glycoprotein IIIa receptor have been shown to be associated with differences in platelet aggregability. The PI(A2) variant of the polymorphism has been reported to be an inherited risk factor for acute coronary events. Although the allele frequency of this polymorphism is well documented in Caucasian populations, studies involving Asian Indians, Malays and Chinese are lacking. We studied 706 random male individuals to determine the genotypic distribution of this polymorphism in Singapore.
METHODS: Male subjects included in this study were drawn from those undergoing routine annual medical examinations offered by their employers. Venous blood was obtained from these patients after an overnight fast and from which genomic DNA was extracted. Genotyping was carried out by polymerase chain reaction (PCR) followed by digestion with restriction enzyme NciI. Personal and family medical history of the subjects were also taken.
RESULTS: The genotype distribution of the individuals studied was in accordance to a population at Hardy Weinberg equilibrium. The frequency of the PI(A2) allele was 0.1, 0.01 and 0.01 in the Indians, Malays and Chinese, respectively. The differences in frequencies of the PI(A2) variant are significant among different ethnic groups (P<0.001 for Indians vs. Chinese and Indians vs. Malays).
CONCLUSIONS: We observed a significantly higher frequency of the PI(A2) allele among Indians relative to the Chinese and Malays in Singapore. The effect of this genotype may partially explain the higher rate of ischaemic heart disease seen among Indians compared to the Chinese and Malay ethnic groups.
ABCA1 gene polymorphisms and their associations with coronary artery disease and plasma lipids in males from three ethnic populations in Singapore

Tan JH, Low PS, Tan YS, Tong MC, Saha N, Yang H, et al.

Hum Genet, 2003 Jul;113(2):106-17.
PMID: 12709788

Mutations in the ATP-binding cassette transporter ABCA1 underlie Tangier disease and familial hypoalphaliproteinemia (FHA), disorders that are characterised by reduced high-density lipoprotein-cholesterol (HDL-C) concentration and cholesterol efflux, and increased coronary artery disease (CAD). We explored if polymorphisms in the ABCA1 gene are associated with CAD and variations in plasma lipid levels, especially HDL-C, and whether the associations may depend on ethnicity. Male cases and controls from the Singapore Chinese, Malay and Indian populations were genotyped for five ABCA1 single nucleotide polymorphisms. Various single-locus frequency distribution differences between cases and controls were detected in different ethnic groups: the promoter -14C>T in Indians, exon 18 M883I in Malays, and 3'-untranslated (UTR) region 8994A>G in Chinese. For the Malay population, certain haplotypes carrying the I825- A (exon 17) and M883- G alleles were more frequent among cases than controls, whereas the converse was true for the alternative configuration of V825- G and I883- A, and this association was reinforced in multi-locus disequilibrium analysis that utilized genotypic data. In the healthy controls, associations were found for -14C>T genotypes with HDL-C in Chinese; 237indelG (5'UTR) with apolipoprotein A1 (apoA1) in Malays and total cholesterol (TC) in Indians; M883I with lipoprotein(a) [Lp(a)] in Malays and apolipoprotein B (apoB) in Chinese; and 8994A>G with Lp(a) in Malays, and TC, low-density lipoprotein-cholesterol (LDL-C) as well as apoB in Indians. While genotype-phenotype associations were not reproduced across populations and loci, V825I and M883I were clearly associated with CAD status in Malays with no effects on HDL-C or apoA1.
Giant Oyster Mushroom Pleurotus giganteus (Agaricomycetes) Enhances Adipocyte Differentiation and Glucose Uptake via Activation of PPARγ and Glucose Transporters 1 and 4 in 3T3-L1 Cells

Paravamsivam P, Heng CK, Malek SN, Sabaratnam V, M RR, Kuppusamy UR

Int J Med Mushrooms, 2016;18(9):821-831.
PMID: 27910773

The edible mushroom Pleurotus giganteus was tested for its effect on adipocyte differentiation and glucose uptake activity in 3T3-L1 cells. The basidiocarps of P. giganteus were soaked in methanol to obtain a crude methanol extract and then fractionated to obtain an ethyl acetate extract. In this study, cell proliferation was measured using an MTT assay, lipid accumulation using an Oil Red O assay, and glucose uptake using a fluorescence glucose uptake assay. Gene expression was measured via real-time polymerase chain reaction analysis with TaqMan primer. Ethyl acetate extract significantly enhanced adipogenic differentiation and glucose uptake in 3T3-L1 adipocytes via the expression of sterol regulatory element-binding protein, peroxisome proliferator-activated receptor γ, and phos-phatidylinositol 3-kinase/Akt. Glucose uptake was facilitated by the highly expressed glucose transporters Glut1 and Glut4. Taken together, these results suggest that P. giganteus ethyl acetate extract has an insulin-sensitizing effect on adipocytes and has potential as an adjuvant for the management of type 2 diabetes.
Association of single nucleotide polymorphism rs6903956 on chromosome 6p24.1 with coronary artery disease and lipid levels in different ethnic groups of the Singaporean population

Tayebi N, Ke T, Foo JN, Friedlander Y, Liu J, Heng CK

Clin Biochem, 2013 Jun;46(9):755-9.
PMID: 23337689 DOI: 10.1016/j.clinbiochem.2013.01.004

A recent genome wide association study in the Chinese population has implicated rs6903956 within the ADTRP gene on chromosome 6p24.1 as a novel susceptibility locus for coronary artery disease (CAD). In this study, we evaluated the association of rs6903956 with CAD in the different ethnic groups of Singaporean population comprising Chinese, Malays and Asian Indians.

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