Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164).
BACKGROUND: Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles.
METHOD: Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results.
RESULTS: We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence.
CONCLUSION: This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.
BACKGROUND: Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4.
METHOD: Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers.
RESULT: The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method.
CONCLUSION: We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.
Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification.
Seventeen single nucleotide polymorphisms (SNPs) have been identified so far, within the beta-2 receptor (beta(2) AR) gene. The presence of so many SNPs within the beta(2) AR gene causes a problem, for those studying beta(2) AR pharmacogenetics, in relation to which SNPs to choose. Most of the work has focused on the three common SNPs within the coding block (alleles 16, 27 and 164) and the techniques developed have been for these three functionally important alleles.
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.
Muntingia calabura L. (family Elaeocarpaceae) has been traditionally used to relieve various pain-related ailments. The present study aimed to determine the antinociceptive activity of methanol extract of M. calabura leaves (MEMC) and to elucidate the possible mechanism of antinociception involved. The in vivo chemicals (acetic acid-induced abdominal constriction and formalin-, capsaicin-, glutamate-, serotonin-induced paw licking test) and thermal (hot plate test) models of nociception were used to evaluate the extract antinociceptive activity. The extract (100, 250, and 500 mg/kg) was administered orally 60 min prior to subjection to the respective test. The results obtained demonstrated that MEMC produced significant (P < 0.05) antinociceptive response in all the chemical- and thermal-induced nociception models, which was reversed after pretreatment with 5 mg/kg naloxone, a non-selective opioid antagonist. Furthermore, pretreatment with L-arginine (a nitric oxide (NO) donor), N(G)-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase (NOS)), methylene blue (MB; an inhibitor of cyclic-guanosine monophosphate (cGMP) pathway), or their combination also caused significant (P < 0.05) change in the intensity of the MEMC antinociception. In conclusion, the MEMC antinociceptive activity involves activation of the peripheral and central mechanisms, and modulation via, partly, the opioid receptors and NO/cGMP pathway.
The genetically polymorphic cytochrome P450 (CYP) 2A6 is the major nicotine-oxidase in humans that may contribute to nicotine dependence and cancer susceptibility. The authors investigated the types and frequencies of CYP2A6 alleles in the three major ethnic groups in Malaysia and CYP2A6*1A, CYP2A6*1B, CYP2A6*1x2, CYP2A6*2, CYP2A6*3, CYP2A6*4, CYP2A6*5, CYP2A6*7, CYP2A6*8 and CYP2A6*10 were determined by allele-specific polymerase chain reaction (PCR) in 270 Malays, 172 Chinese and 174 Indians. Except for CYP2A6*2 and *3 that were not detected in the Malays and Chinese, all the other alleles were detected. Frequencies for the CYP2A6*4 allele were 7, 5 and 2%, respectively, in Malays, Chinese and Indians. A statistically significant high frequency of the duplicated CYP2A6*1x2 allele occurred among Chinese. Among Malays and Chinese, the most common allele was CYP2A6*1B, but it was CYP2A6*1A among Indians. These ethnic difference in frequencies suggested that further studies are required to investigate the implications on diseases such as cancer and smoking behaviour among these major ethnic groups in Malaysia.
Genetic polymorphisms of CYP2C9 among different populations in different geographical regions could be different. CYP2C9 has been reported to be the enzyme responsible for the metabolism of many drugs, including warfarin and other drugs with a narrow therapeutic index. Realising the importance of inter-individual differences in the genetic profile in determining the outcome of a drug therapy, this study was conducted to explore the types and frequencies of CYP2C9 alleles in healthy and warfarin-treated Malays and Chinese, the two major ethnic groups in Malaysia. We aimed to evaluate the prevalence of the types and frequencies of common CYP2C9 alleles (*1, *2, *3 and *4) among the healthy unrelated individuals and diseased patients prescribed with warfarin.
Although they originated from China, Malays have undergone a lot of intermarriages. A study suggested that CYP2D6 poor metabolism (PM) phenotype was more common in Malays compared to Chinese. CYP2D6 is highly polymorphic and is involved in the metabolism of many drugs and has been implicated in some environmentally-induced diseases. It is therefore useful to further study this polymorphism in Malays.
Despite concerns about adverse effects, chloramphenicol (CMC) continues to be used in certain situations and, due to its low therapeutic index and variable pharmacokinetics, therapeutic drug monitoring (TDM) is often recommended. At our centre, CMC finds applications in typhoid and meningitis and TDM is routinely performed. Elsewhere in Malaysia, however, CMC is used without TDM. We therefore decided to evaluate our TDM for CMC in relation to its roles in CMC therapy in children, who constitute most of our patients. Our objective was also to develop strategies to improve our TDM for CMC use. Data were collected from 168 children given CMC for various indications and monitored by the TDM service. Plasma CMC was determined by HPLC and used to adjust doses to maintain concentrations within a range of 10-25 micrograms/ml. Outcomes measured included daily temperatures and haematological indices. Daily doses and plasma CMC varied greatly. Doses averaged 40.5 mg/kg for neonates and 75.5 for older children. Average peak concentrations were therapeutic in 60% and trough in 42%. Average duration of fever was 6.3 days and it was unaffected by plasma CMC. Typhoid was eradicated in 97% but nine children with other diagnoses died. Side-effects were confined to mild reversible haematological abnormalities which developed in 11% of children at plasma concentrations which tended to be high. We conclude that CMC remains useful in children with typhoid. Its use for other indications, however, should be reviewed. Routine TDM for CMC is probably not warranted, at least until a clearer role is defined by well designed prospective studies.
Testing for cytochrome P450-2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) variant alleles is recommended by the FDA for dosing of warfarin. However, dose prediction models derived from data obtained in one population may not be applicable to another. We therefore studied the impact of genetic polymorphisms of CYP2C9 and VKORC1 on warfarin dose requirement in Malaysia.
This is the first documentation of the complete mitochondrial genome sequence of the Malaysian Mahseer, Tor tambroides. The 16,690 bp mitogenome with GenBank accession number JX444718 contains 13 protein genes, 22 tRNAs, two rRNAs, and a noncoding control region (D-loop) as is typical of most vertebrates. The phylogenomic reconstruction of this newly generated data with 21 Cypriniformes GenBank accession ID concurs with the recognized status of T. tambroides within the subfamily Cyprininae. This is in agreement with previous hypotheses based on morphological and partial mitochondrial analyses.
Klebsiella pneumoniae PR04 was isolated from a patient hospitalized in Malaysia. The draft genome sequence of K. pneumoniae PR04 shows differences compared to the reference sequences of K. pneumoniae strains MGH 78578 and NTUH-K2044 in terms of their genomic structures.
Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium that was first recognized as a causative agent of bovine mastitis. S. agalactiae has subsequently emerged as a significant cause of human diseases. Here, we report the draft genome sequence of S. agalactiae PR06, which was isolated from a septicemic patient in a local hospital in Malaysia.
We report the annotated genome sequence of a clinical isolate, Mycobacterium tuberculosis strain PR05, which was isolated from the human cerebrospinal fluid of a patient diagnosed with tuberculosis.
Expanding the scope of pharmacogenomic research by including multiple global populations is integral to building robust evidence for its clinical translation. Deep whole-genome sequencing of diverse ethnic populations provides a unique opportunity to study rare and common pharmacogenomic markers that often vary in frequency across populations. In this study, we aim to build a diverse map of pharmacogenetic variants in South East Asian (SEA) Malay population using deep whole-genome sequences of 100 healthy SEA Malay individuals. We investigated the allelic diversity of potentially deleterious pharmacogenomic variants in SEA Malay population. Our analysis revealed 227 common and 466 rare potentially functional single nucleotide variants (SNVs) in 437 pharmacogenomic genes involved in drug metabolism, transport and target genes, including 74 novel variants. This study has created one of the most comprehensive maps of pharmacogenetic markers in any population from whole genomes and will hugely benefit pharmacogenomic investigations and drug dosage recommendations in SEA Malays.
CYP2C8 is genetically polymorphic. Four variants, CYP2C8*2, CYP2C8*3, CYP2C8*4 and CYP2C8*5, which contain mutations in the coding regions have been reported to exhibit different enzyme activity as compared with CYP2C8*1.
Although Malaysian Chinese share an origin with the mainland Chinese, their evolution has been influenced by intermarriages. With a gene such as CYP2D6, which is highly polymorphic, it is expected that the Malaysian Chinese would exhibit a polymorphism profile different from those of the Chinese populations in other geographical locations.