Di-nucleotide microsatellites were isolated from a genomic library of a tropical tree species, Dryobalanops lanceolata, in Sarawak, for the purpose of using them as hypervariable genetic markers to study the pollen-mediated gene flow. Among 1600 recombinant clones, in total 20 clones gave positive signals when hybridized with oligonucleotides with the three different repeat motifs, GT, CA and CT. Estimations of abundance of (GT)n/(CA)n and (GA)n/(CT)n dinucleotide repeats in D. lanceolata genome revealed to be one in every 84 kb and 80 kb, respectively. Among six sequenced microsatellite loci, one was selected to synthesize PCR primers to amplify the microsatellite. PCR product size of the locus was variable among different individuals, which is attributed to the different number of di-nucleotide repeats. The same microsatellite genotype was detected in the trunk and canopy of a single large tree, indicating the utility of trunk tissue as the source of DNA for the population genetic study of tropical tree species, the canopy of which is usually difficult to approach.
Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disease characterized by neoplasia of the parathyroid glands, anterior pituitary and endocrine pancreas, is rarely reported in Asian populations. The MEN1 gene, mapped to chromosome 11q13 but yet to be cloned, has been found to be homogeneous in Caucasian populations through linkage analysis. Here, two previously unreported Asian kindreds with MEN1 are described; linkage analysis using microsatellite polymorphic markers in the MEN1 region was carried out. The first kindred, of Mongolian-Chinese origin, is a multigeneration family with over 150 living members, eight of whom are affected to date. The second kindred is of Chinese origin consisting of four affected members. Linkage to chromosome 11q13 was confirmed in both kindreds, supporting evidence for genetic homogeneity. A recombination in the larger kindred localizes the gene distal to marker D11S956, consistent with its placement from previous studies. We also show that it is feasible to use these markers for predictive testing, as four gene carriers were detected in 13 family members with unknown disease status in the first kindred.
Discriminatory typing methods are invaluable in the investigation of outbreaks of infectious diseases. Single primers were used to generate randomly amplified polymorphic DNA (RAPD) profiles from Klebsiella pneumoniae isolates of various serotype and K. pneumoniae isolates from cases of sepsis at a Malaysian hospital and two English hospitals. RAPD profiles of acceptable reproducibility, a maximum of three minor band variations, were produced using a rapid DNA extraction method. RAPD typing of K. pneumoniae was shown to be as discriminatory as restriction fragment length polymorphism analysis using pulsed field gel electrophoresis yet quicker and less costly. The findings suggest that RAPD typing may be a useful tool for the epidemiological typing of K. pneumoniae.
Members of the Semai group of Orang Asli ('aborigines') in peninsular Malaysia were examined for apolipoprotein E (apo E) variants in relation to plasma total cholesterol (TC), high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDLC), triglycerides (TG), apolipoprotein AI and apolipoprotein B (apo B). The e2 and e4 alleles were found to be higher than in most other groups as reported. The sample as a whole was normotriglyceridaemic (mean plasma TG, 1.5 mmol/l) and very markedly hypocholesterolaemic (mean plasma TC 1.7 mmol/l). The distribution of apo E variants was not related to any of the plasma lipids or apolipoprotein fractions using results from all subjects, but if a distinctly hypertriglyceridaemic sub-section was omitted (TG > 1.7 mmol/l) then apo E variants were determinants of plasma TC, LDLC, and apo B concentrations, the lower values of these being associated with the 2-2 and 2-3 genotypes, and the higher with 3-4, and 4-4.
We report the fact that D. albomicans invaded into Shanghai suddenly in the autumn of 1991. Using 9 restriction enzymes, we analyse the RFLPs of mitochondrial DNA of 29 isofemale lines belonging to 4 populations of Shanghai, Jiading, Qinpu and Nanhui. We find that all 29 haplotypes are different from each other. Comparing with the populations of Canton, Kunming, Sanhutan (Taiwan), Sumoto (Japan), and Kuala Lumper (Malaysia), we come to the conclusion that D. albomicans caught in Shanghai and areas nearby is from a few of places in the south of China-mainland. This conclusion agrees with the viewpoint that this species is on the speciation stage of migration towards north. We also discuss the mtDNA polymorphism within the species.
A population genetic study was undertaken to provide gene frequency data on the additional blood genetic markers in the Semai and to estimate the genetic relations between the Semai and their neighboring and linguistically related populations by genetic distance and principal components analyses. Altogether 10 polymorphic and 7 monomorphic blood genetic markers (plasma proteins and red cell enzymes) were studied in a group of 349 Senoi Semai from 11 aboriginal settlements (villages) in the Pahang State of western Malaysia. Both the red cell glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) loci reveal the presence of polymorphic frequencies of a nondeficient slow allele at the G6PD locus and a fast allele at the PGD locus. The Semai are characterized by high prevalences of ahaptoglobinemia and G6PD deficiency, high frequencies of HP*1, HB*E, RH*R1, ACP*C, GLO1*1, PGM1*2+, and GC*1F and corresponding low frequencies of ABO*A, HbCoSp, HB*B0, TF*D, CHI, and GC*2. Genetic distance analyses by both cluster and principal components models were performed between the Semai and 14 other populations (Malay; Javanese; Khmer; Veddah; Tamils of Malaysia, Sri Lanka, and India; Sinhalese; Oraon; Toda and Irula of India; Chinese; Japanese; Koreans) on the basis of 30 alleles at 7 polymorphic loci. A more detailed analysis using 53 alleles at 13 polymorphic loci with 10 populations was carried out. Both analyses give genetic evidence of a close relationship between the Semai and the Khmer of Cambodia. Furthermore, the Semai are more closely related to the Javanese than to their close neighbors--the Malay, Chinese, and Tamil Indians. There is no evidence for close genetic relationship between the Semai and the Veddah or other Indian tribes. The evidence fits well with the linguistic relationship of the Semai with the Mon-Khmer branch of the Austro-Asiatic language family.
Glutathione S-transferase-theta (GSTT1) is subject to a genetic polymorphism where approximately 50% of a Caucasian population are homozygous for the null allele. Because of the possible association of the polymorphism with increased cancer risk in individuals, we genotyped by polymerase chain reaction 187 normal Chinese, 167 normal Malays and 152 normal Indians from Singapore and Malaysia. The proportion of Chinese, Malays and Indians with the null genotype were 58%, 38% and 16% respectively and mirrored previously reported frequencies of the GSTM1 null genotype in these populations. The frequency of the combined GSTM1 and GSTT1 null genotypes among Chinese, Malays and Indians were 37%, 22% and 5% respectively. The similarity with predicted frequencies indicated no interaction between the two genetic polymorphisms.
The distribution of two common DNA polymorphisms (5' untranslated exon 1 and intron 5-DdeI) of the antithrombin III (ATIII) gene was studied in three ethnic groups in Singapore: 251 Chinese, 221 Dravidian Indians and 102 Malays. The polymorphisms were identified by the polymerase chain reaction and size fractionation in agarose gels. The 5' untranslated to exon 1 polymorphism is a length polymorphism while the intron 5 polymorphism is a restriction site (DdeI) polymorphism. The frequency of the short fragment (S) of the 5' to exon 1 length polymorphism of the ATIII gene was found to be 0.37 in the Chinese, 0.54 in the Malays and 0.65 in the Dravidian Indians. For the Chinese, this was significantly lower compared to the Caucasians and Indians (p < 0.0001) and the Malays (p < 0.01). On the other hand, the frequencies of DdeI+ did not vary significantly among these three populations (p > 0.05). The distribution of different genotypes at these two loci of the ATIII gene was in Hardy-Weinberg equilibrium in all three ethnic groups. A strong linkage disequilibrium between these two polymorphisms was observed in all the ethnic groups and the estimated correlation coefficient (delta) was 0.42 in the Chinese (p < 0.001), 0.61 in the Dravidian Indians (p < 0.001) and 0.43 in the Malays (p < 0.001). The frequencies of haplotype S+, L+ and L- were, respectively, 0.37, 0.40 and 0.23 in the Chinese, 0.65, 0.18 and 0.16 in the Dravidian Indians and 0.54, 0.37 and 0.09 in the Malays.(ABSTRACT TRUNCATED AT 250 WORDS)
A study was initiated to amplify by polymerase chain reaction (PCR), a short factor VIII gene fragment containing the Bcl I restriction site from hemophilia patients using published primer sequences. Preliminary findings indicated that the resulting fragment is 142 bp long. This fragment, when digested with Bcl I restriction enzyme produced two fragments, 99 bp and 43 bp in length. Polymorphism in the Bcl I region can be used to detect carrier state in the family members of the hemophiliacs.
We have examined 56 unrelated individuals from Malaysian aborigines for their DNA polymorphism of the HLA-B gene by sequence specific oligonucleotide probe (SSO) method. Using the SSO hybridization, we found that one specific DNA allele with a B*1513 like pattern of epitope combination (ECB1513) was dominant among the Melayu Asli (Af = 41.9%) and the Senoi (Af = 24%). To determine the nucleotide sequences of ECB1513, a DNA fragment spanning from the beginning of exon 1 to the middle of exon 4 of the HLA-B gene was amplified by polymerase chain reaction (PCR) from two ECB1513 positive individuals, and the PCR products were cloned and sequenced. This sequencing analysis confirmed that ECB1513 was identical to HLA-B*1513 in exon 1, 2, 3, and 4. Amino acid sequence of this major allele, HLA-B*1513, in the aborigines especially around the peptide binding groove (B and F pockets), was compared with that of African B*5301 that had been suggested to confer resistance to malaria infection in Africa. The amino acid residues composing of the F pocket were completely identical in B*1513 and B*5301. These observations suggest that a common environmental factor, the malaria infection, might have independently enhanced the selection of functional change in the polymorphic portion of HLA-B gene in Africa and in South-East Asia.
Five polymorphisms in the C6 and C7 genes have been investigated in seven ethnic groups. The allele frequencies are broadly similar in most groups except C7 M/N which is monomorphic in our group of Africans, and C6 MspI and C7 S367T where the allele frequencies in African and Cape Coloured subjects are very different from the other ethnic groups. There is very little allelic association except between C6 A/B and C6 MspI. Seventeen of the 32 possible haplotypes have been observed, suggesting that much recombination has taken place. We describe a new method for the investigation of the MspI RFLP located in intron 3 of C6 (approximately 3 kbp 3' from exon 3 and 1.5 kbp 5' from exon 4) and its molecular basis, together with an improved method for the isolation of DNA from stored serum.
The Arg-to-Trp substitution at codon 3500 in the apolipoprotein (apo) B-100 gene is established as a cause of familial defective apo B-100 (FDB), a functional mutation, resulting in reduced LDL receptor binding and manifest hypercholesterolemia. In a search for similar mutations in 163 Malaysians, we screened the putative receptor-binding region (codons 3456-3553) of the apo B-100 gene by PCR amplification and denaturing gradient-gel electrophoresis. Four single-base mutations were detected and confirmed by DNA sequencing. Two females, a Chinese and a Malay, had the same CGG3500-->TGG mutation, resulting in an Arg3500-to-Trp substitution. This is the second published report of such an independent mutation involving the same codon as the established Arg3500-to-Gln mutation. The two other mutations detected, CTT3517-->CTG and GCC3527-->GCT, resulted in degenerate codons with no amino acid substitutions. All four mutations were associated with a unique apo B haplotype, different from those found in Caucasian FDB patients but concurring with that previously reported for two other Asians with FDB.
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.
The xenobiotic metabolizing enzymes N-acetyltransferases (NATs) are important for the biotransformation and/or bioactivation of drugs and carcinogens. NATs are coded for in humans by two distinct genes, designated NAT1 and NAT2. NAT1, which was originally thought to be monomorphic, was recently reported to exhibit variation in human populations. Recent studies suggested that a genetic polymorphism of NAT1 may be associated with colorectal cancer risk. The distributions of NAT1 allele and genotype frequencies in unrelated individuals among Indian (n = 140), Malay (n = 122) and Chinese (n = 181) populations in Singapore were characterized by polymerase chain reaction-restriction fragment length polymorphism and allele-specific-polymerase chain reaction. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Indians were 0.3, 0.51, 0.17 and 0.02, respectively. The corresponding NAT1 allelic frequencies in Malays were 0.29, 0.30, 0.39 and 0.02, respectively, and were similar to those in Chinese in the region. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Chinese were 0.33, 0.35, 0.30 and 0.02, respectively. These findings are of importance for the determination of cancer risk in these populations. In addition, nucleotide changes at positions 350-351 (GG to CC) and 497-499 (GGG to CCC) of the NAT1 gene were not found in the alleles of the populations studied.
Nine simple sequence repeat (SSR) markers were developed from Shorea curtisii using two different methods. One SSR locus was isolated by the commonly used method of screening by colony hybridization, and the other eight loci were isolated by a vectorette PCR method. Primer pairs were designed based on the sequences of all these SSR loci. Analysis of 40 individuals of S. curtisii from natural forest in Malaysia revealed that all SSR loci were polymorphic. Four SSR markers, Shc01, Shc04, Shc07 and Shc09, were highly polymorphic. We have also tested the applicability of these SSR printers to other species of Dipterocarpaceae using PCR amplification. Because the flanking region sequences of the S. curtisii SSRs were well conserved within this family, the SSR primers for S. curtisii can be applied to almost all species of Dipterocarpaceae.
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.
The neutrophil antigen (NA)1 and 2 is coded by two recognized allelic forms of Fc gamma receptor IIIB (FcgammaRIIIB). FcgammaRIIIb is a low affinity receptor and preferentially removes immune complexes from the circulation. Systemic lupus erythematosus (SLE) is an autoimmune and polygenic disorder characterized by accumulation of autoimmune complexes. The majority of SLE patients in our medical center are of Chinese ethnicity, followed by Malay and Indian. Recently, studies have focussed on the Fc receptors in different ethnic groups and their relation to SLE. We chose to study the gene distribution of this receptor in the Chinese and Malays population in Malaysia. We designed a polymerase chain reaction allele specific primers (PCR-ASP) method to distinguish the two allelic forms. Genomic DNA was isolated from the peripheral blood of 183 Chinese and 55 Malays SLE patients as well as 100 Chinese and 50 Malays healthy controls. Genotyping of Chinese SLE patients revealed that the gene frequencies for FcgammaRIIIB-NA1 and FcgammaRIIIB-NA2 were 0.648 and 0.347, while in the ethnically matched healthy controls they were 0.68 and 0.32, respectively. One out of the 183 Chinese SLE patients was identified as a NA-null due to the absence of PCR product for both alleles. The FcgammaRIIIB-NA1 and FcgammaRIIIB-NA2 allele frequencies for both the Malays SLE and healthy controls were 0.62 and 0.38.
Apolipoprotein E (APOE = gene, apoE = protein) plays a central role in plasma lipoprotein metabolism and in lipid transport within tissues. The APOE shows a genetic polymorphism determined by three common alleles, APOE*2, APOE*3, APOE*4 and the product of the three alleles differs in several functional properties. APOE is involved in the development of certain pathological conditions. In particular, the APOE*4 allele is a risk factor for susceptibility to coronary artery disease (CAD) and Alzheimer's Disease (AD). In the present study we analyzed the APOE allele distribution in the world. The APOE*3 is the most frequent in all the human groups, especially in populations with a long-established agricultural economy like those of the Mediterranean basin (0.849-0.898). The frequency of APOE*4, the ancestral allele, remains higher in populations like Pygmies (0.407) and Khoi San (0.370), aborigines of Malaysia (0.240) and Australia (0.260), Papuans (0.368), some Native Americans (0.280), and Lapps (0.310) where an economy of foraging still exists, or food supply is (or was until the recent past) scarce and sporadically available. The APOE*2 frequency fluctuates with no apparent trend (0.145-0.02) and is absent in Native Americans. We suggest that the APOE*4, based on some functional properties it has and on its distribution among human populations, could be identified as a 'thrifty' allele. The exposure of APOE*4 to the contemporary environmental conditions (Western diet, longer lifespans) could have rendered it a susceptibility allele for CAD and AD. The absence of the association of APOE*4 with CAD and AD in Sub-Saharan Africans, and its presence in African Americans, seems to confirm this hypothesis.
SLE is an autoimmune and polygenic disorder characterized by an accumulation and deposition of immune complexes. Several studies have indicated differential impact of FcgammaR polymorphism genotypes in different ethnic groups studied. The Fc receptor for IgG class IIA gene (FcgammaRIIA) occurs in two allelic forms. The allele FcgammaRIIA-H131 encodes a receptor with a histidine at the 131 amino acid position; the other allele FcgammaRIIA-R131 encodes an arginine. This polymorphism is believed to determine the affinity of the receptor for hIgG2 in immune complexes. FcgammaRIIA-H131 has a higher capacity for hIgG2 compared to FcgammaRIIA-R131 as measured by in vitro studies of insoluble immune complex clearance. We have investigated the polymorphism for FcgammaRIIA using a novel polymerase chain reaction-allele specific primer (PCR-ASP) method designed specifically to distinguish the two allelic forms. Our studies were based on 175 Chinese and 50 Malays SLE patients as well as 108 and 50 ethnically matched healthy controls for the respective groups. Analysis of the data (chi2 test with Yates correction factors and odds ratios) revealed that there were no significant differences between SLE patients and controls. We have not found evidence of a protective effect conferred by FcgammaRIIA-H131 in the ethnic groups studied.