OBJECTIVES: Recently, several genome-wide association studies have demonstrated a cumulative association of 5 polymorphic variants in chromosomes 8q24 and 17q with prostate cancer (CaP) risk in Caucasians, particularly those harboring aggressive clinicopathologic characteristics. The purpose of this study was to evaluate the influence of these variants on CaP susceptibility in Singaporean Asian men.
MATERIALS AND METHODS: We performed a case-control study in 289 Chinese CaP patients and 412 healthy subjects (144 Chinese, 134 Malays, and 134 Indians), and examined the association of the 5 single nucleotide polymorphisms (SNPs) with CaP.
RESULTS: In the healthy subjects, rs16901979 A-allele frequency was highest amongst Chinese (0.32) compared with Malays (0.13; P < 0.0001) or Indians (0.09; P < 0.0001); rs6983267 G-allele was highest in Indians (0.51) compared with Chinese (0.42; P = 0.041) or Malays (0.43; P = 0.077); whereas rs1859962 G-allele frequency was highest amongst Indians (0.56) compared with Chinese (0.40; P = 0.0002) or Malays (0.38; P < 0.0001). Individuals with the rs4430796 TT genotype were at increased CaP risk in the Chinese via a recessive model (odds ratios (OR) = 1.56, 95% CI = 1.04-2.33). Significant associations were observed for rs4430796 TT with Gleason scores of ≥ 7 (OR = 1.76, 95% CI = 1.14-2.73) and prostate-specific antigen (PSA) levels of ≥ 10 ng/ml at diagnosis (OR = 1.63, 95% CI = 1.01-2.63), as well as for rs6983267 GG with stage 3-4 CaPs (OR = 1.91, 95% CI = 1.01-3.61). A cumulative gene interaction influence on disease risk, which approximately doubled for individuals with at least 2 susceptibility genotypes, was also identified (OR = 2.18, 95% CI = 1.10-4.32).
CONCLUSIONS: This exploratory analysis suggests that the 5 genetic variants previously described may contribute to prostate cancer risk in Singaporean men.
KEYWORDS: Cancer; Ethnicity; Gleason; Pharmacogenetics; Polymorphism; Prostate
The application of PCR technique in genetic screening was demonstrated using the genetic materials from buccal cells of the students in the class. Two factors were taken into consideration when designing the experiments. The DNA region to be amplified should not be associated with any disease state. This is to eliminate any emotional and ethical problems associated with the experiments. In this practical, the presence and absence of a 38 bp sequence in the intron of COLIA2 gene were studied. The students were also shown on how to analyse the presence of homozygous and heterozygous alleles and the genetic variations that might be observed in the different ethnic groups of students. Another factor was the time taken to complete the experiment. Our experience showed that this experiment would take at least six hours to obtain and analyse the results. It is therefore suitable to be used in class teaching.
Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.
Exercise has been suggested as the best and the most affordable way for managing blood pressure. The insertion/ deletion of angiotensin I-converting enzyme (ACE) I/D gene polymorphism had been reported to be linked with sev- eral diseases such as hypertension and diabetic nephropathy. Several studies showed that blood pressure response to exercise training for health management also vary among individuals with different genotypes of ACE I/D gene poly- morphism. A study of 9 months of endurance exercise training at 75 to 85 % of VO2max showed that the decrease of resting blood pressure in I allele carriers wass greater than D allele carriers. In contrast, other study discovered that adult women with D allele had greater reduction in resting blood pressure than those with I allele, following a 12-week combined aerobic and resistance exercise training. Despite the inconsistencies of some findings, it has remained unknown if the ACE I/D gene polymorphism would also influence blood pressure response to isometric handgrip training that had been found to be superior to the dynamic resistance exercise training in controlling and preventing high blood pressure. Thus, this article was to review the literature on ACE I/D gene polymorphism and blood pressure response to exercise training that could serve as the basis for future research to identify individuals who will lower resting blood pressure the most with exercise training program for health management.
Hb Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a rare hemoglobin (Hb) variant due to a mutation at codon 59 of the α2- or α1-globin gene resulting in a glycine to aspartic acid substitution. Two siblings with a unique coinheritance of Hb Adana and Hb Constant Spring (Hb CS, α142, Term→Gln, TAA>CAA; HBA2: c.427 T>C) (α(codon 59)α/α(CS)α), were compared phenotypically with another two siblings carrying the Hb Adana mutation and a 3.7 kb deletion (α(codon 59)α/-α(3.7)). Although they all had α-thalassemia intermedia (α-TI), the former were clinically more severe than the latter. The first pair of siblings presented at a much younger age than the second pair and showed lower Hb levels and significant extramedullay hemopoiesis. Another case of a hydropic fetus as a result of Hb H/Hb Adana is also described. Their clinical phenotypes and hematological parameters are all presented for comparison.
Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10(-33)), we observed one SNP showing significant association to POAG (CDC7-TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10(-8)). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis.
To identify a gene(s) susceptible to nasopharyngeal carcinoma (NPC), we carried out a genome-wide association study (GWAS) through genotyping of more than 500,000 tag single-nucleotide polymorphisms (SNPs), using an initial sample set of 111 unrelated NPC patients and 260 controls of a Malaysian Chinese population. We further evaluated the top 200 SNPs showing the smallest P-values, using a replication sample set that consisted of 168 cases and 252 controls. The combined analysis of the two sets of samples found an SNP in intron 3 of the ITGA9 (integrin-alpha 9) gene, rs2212020, to be strongly associated with NPC (P=8.27 x 10(-7), odds ratio (OR)=2.24, 95% confidence intervals (CI)=1.59-3.15). The gene is located at 3p21 which is commonly deleted in NPC cells. We subsequently genotyped additional 19 tag SNPs within a 40-kb linkage disequilibrium (LD) block surrounding this landmark SNP. Among them, SNP rs189897 showed the strongest association with a P-value of 6.85 x 10(-8) (OR=3.18, 95% CI=1.94-5.21), suggesting that a genetic variation(s) in ITGA9 may influence susceptibility to NPC in the Malaysian Chinese population.
The adiponutrin (PNPLA3) rs738409 polymorphism has been found to be associated with susceptibility to non-alcoholic fatty liver disease (NAFLD) in various cohorts. We further investigated the association of this polymorphism with non-alcoholic steatohepatitis (NASH) severity and with histological features of NAFLD. A total of 144 biopsy-proven NAFLD patients and 198 controls were genotyped for PNPLA3 gene polymorphism (rs738409 C>G). The biopsy specimens were histologically graded by a qualified pathologist. We observed an association of G allele with susceptibility to NAFLD in the pooled subjects (OR 2.34, 95% CI 1.69-3.24, p < 0.0001), and following stratification, in each of the three ethnic subgroups, namely Chinese, Indian and Malay (OR 1.94, 95% CI 1.12-3.37, p = 0.018; OR 3.51, 95% CI 1.69-7.26, p = 0.001 and OR 2.05, 95% CI 1.25-3.35, p = 0.005, respectively). The G allele is associated with susceptibility to NASH (OR 2.64, 95% CI 1.85-3.75, p < 0.0001), with NASH severity (OR 1.85, 95% CI 1.05-3.26, p = 0.035) and with presence of fibrosis (OR 1.95, 95% CI 1.17-3.26, p = 0.013) but not with simple steatosis nor with other histological parameters. Although the serum triglyceride level is significantly higher in NAFLD patients compared to controls, the G allele is associated with decreased level of triglycerides (p = 0.029) in the NAFLD patients. Overall, the rs738409 G allele is associated with severity of NASH and occurrence of fibrosis in patients with NAFLD.
A novel human leukocyte antigen-B allele officially named B*3589, was found in an indigenous individual of Jehai ethnicity when sequencing was performed to investigate human genome variation in a research project. B*3589 differs form B*3505 in a point mutation at codon 169 (CGC to TGC) resulting in an amino acid change from Arg to Cys.
Fragile X Syndrome (FXS) is the most common form of inherited mental retardation in men. It is caused by abnormalities in the FMR1 gene that are associated with CGG repeat expansion and the hypermethylation status of its promoter. Methylated alleles lead to transcriptional inhibition and consequent loss of Fragile X Mental Retardation Protein. Chemical modification of cytosine to uracil by bisulfite treatment has proved to be an attractive method for DNA methylation studies that precludes labor-intensive Southern blot analysis, which is the gold standard test for FXS. In this report, bisulfite-treated DNA samples were amplified using real-time multiplex methylation-specific polymerase chain reaction followed by melting curve analysis. Our results show that all control samples with known CGG repeat numbers and methylation statuses were correctly diagnosed. The samples from 43 male patients were also successfully diagnosed, which were in complete agreement with the results of Southern blotting. By such means, 39 patients were found to have an unmethylated allele; 3, a fully mutated allele; and 1, both methylated and unmethylated alleles, thus implying a diagnosis of mosaicism. In conclusion, we find our method to be convenient for screening a large number of male patients with FXS, because it is rapid and easy to perform, especially when there is a low quantity of DNA that must be sensitively and accurately assayed.
The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods.
Ankylosing spondylitis (AS) is a chronic inflammatory disorder with predilection for the axial skeleton, leading to progressive restricted mobility and deformity of the spine. The fundamental mechanism involves autoimmunity orchestrated by T cells. Similar to other rheumatic diseases, the complex interplay of cytokines such as tumour necrosis factor alpha, interleukin-6 (IL 6) and interleukin-10 (IL 10) has been implicated in the pathogenesis of the disease. Despite extensive research over the past decades, the treatment options for AS, are limited. Non steroidal antiinflammatory drugs are the first line of therapy, whereas anti TNF drugs are administered for refractory cases which fail to respond to the treatment. There have been conflicting views on the correlation of IL 6 with disease activity in AS. As such, the debate on the role of anti IL6 in AS is still ongoing. Anti IL 6 such as tocilizumab and siltuximab have proven efficacy based on the large randomized controlled trials. The Food and Drug Administration (FDA) has approved these drugs for treating rheumatoid arthritis and systemic juvenile idiopathic arthritis. Researchers have adventurously experimented anti IL 6 therapy in AS but the conclusions made were not consolidated into international guidelines or consensus statement for clinical practice. In the present review, we explore the role of anti IL6 in the treatment of AS based on the cumulative evidence over recent years.
Urinary schistosomiasis is caused by the digenetic trematode Schistosoma haematobium, characterized by accumulation of eggs in the genitourinary tract. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) can play an important role in parasitic infection due to its major role as a negative regulator of T-cell activation and proliferation. This study was performed in patients with schistosomiasis and healthy controls to analyze the allele and genotype frequencies of four CTLA-4 gene polymorphisms. The CTLA-4 gene was amplified using Taqman real-time polymerase chain reaction, and allele and genotypes of 49 patients with schistosomiasis were analyzed using allelic discrimination analysis followed by subsequent direct sequencing. The results were compared with healthy control subjects. The frequencies of CTLA-4 rs733618 A allele at position -1722 (p=0.001), rs11571316 C allele at position -1577 (p<0.001), and rs231775 A allele at position +49 (p=0.002) in the patient group were significantly higher than the control group. The rs733618 AA genotype (p=0.001), rs11571316 CC genotype (p<0.001), and rs231775 AA genotype (p=0.007) were also significantly overrepresented. Meanwhile, rs733618 AG genotype (p=0.001), rs11571316 CT genotype (p=0.02), and rs231775 GG genotype (p=0.029) were significantly decreased in the patients with schistosomiasis, as compared with the controls. No significant difference was observed in both allele and genotype of rs16841252. The results of this study suggest that the rs733618, rs11571316, and rs231775 polymorphisms in the CTLA-4 gene may influence susceptibility to schistosomiasis infection in the Gabonese children.
The genetic relationship between any two populations is a function of the differences between them in allele frequencies, with this relationship commonly expressed in terms of a genetic distance. For example if the two populations are homozygous for different alleles at a particular locus, the distance is the maximum possible whereas if the allele frequencies in the two populations are identical, the distance is zero. The estimated genetic distances may vary among loci. Thus, the most accurate measures of relationships will be obtained by averaging many loci (Nei, 1978). In plant and animal breeding, it is expected that a cross between two individuals originating from two populations with a large genetic distance between them will result in maximum heterosis or hybrid vigour. Many estimates of genetic distances are available in the literature but Nei’s standard genetic distance, D, (Nei, 1972, 1978) had been extensively used in studies of human, animal and plant varieties, races, breeds, strains, populations, species and genera.
Fanconi Anemia (FA) is an autosomal recessive syndrome characterized by congenital abnormalities, progressive bone marrow failure and Fanconi anemia complementation group A (FANCA) is also a potential breast and ovarian cancer susceptibility gene. A novel allele with tandem duplication of 13 base pair sequence in promoter region was identified. To investigate whether the 13 base pair sequence of tandem duplication in promoter region of the FANCA gene is of high penetrance in patients with breast cancer and to determine if the presence of the duplicated allele was associated with an altered risk of breast cancer, the present study screened DNA in blood samples from 304 breast cancer patients and 295 normal individuals as controls. The duplication allele had a frequency of 35.4 and 21.2% in patients with breast cancer and normal controls, respectively. There was a significant increase in the frequency of the duplication allele in patients with familial breast cancer compared with controls (45.1%, P=0.001). Furthermore, the estimated risk of breast cancer in individuals with a homozygote [odds ratio (OR), 4.093; 95% confidence intervals (CI), 1.957‑8.561] or heterozygote duplicated genotype (OR, 3.315; 95% CI, 1.996‑5.506) was higher compared with the corresponding normal homozygote genotype. In conclusion, the present study indicated that the higher the frequency of the duplicated allele, the higher the risk of breast cancer. To the best of our knowledge, the present study is the first to report FANCA gene duplication in patients with breast cancer.
Over the last few decades, the use of molecular markers has played an increasing role in rice breeding and genetics. Of the different types of molecular markers, microsatellites have been utilized most extensively, because they can be readily amplified by PCR and the large amount of allelic variation at each locus. Microsatellites are also known as simple sequence repeats (SSR), and they are typically composed of 1-6 nucleotide repeats. These markers are abundant, distributed throughout the genome and are highly polymorphic compared with other genetic markers, as well as being species-specific and co-dominant. For these reasons, they have become increasingly important genetic markers in rice breeding programs. The evolution of new biotypes of pests and diseases as well as the pressures of climate change pose serious challenges to rice breeders, who would like to increase rice production by introducing resistance to multiple biotic and abiotic stresses. Recent advances in rice genomics have now made it possible to identify and map a number of genes through linkage to existing DNA markers. Among the more noteworthy examples of genes that have been tightly linked to molecular markers in rice are those that confer resistance or tolerance to blast. Therefore, in combination with conventional breeding approaches, marker-assisted selection (MAS) can be used to monitor the presence or lack of these genes in breeding populations. For example, marker-assisted backcross breeding has been used to integrate important genes with significant biological effects into a number of commonly grown rice varieties. The use of cost-effective, finely mapped microsatellite markers and MAS strategies should provide opportunities for breeders to develop high-yield, blast resistance rice cultivars. The aim of this review is to summarize the current knowledge concerning the linkage of microsatellite markers to rice blast resistance genes, as well as to explore the use of MAS in rice breeding programs aimed at improving blast resistance in this species. We also discuss the various advantages, disadvantages and uses of microsatellite markers relative to other molecular marker types.
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.
The aim of the present study was to investigate the association of C4 gene mutations with systemic lupus erythematosus, in 130 Malaysian SLE patients and 130 healthy controls. Generally, various PCR approaches were used to screen the mutations of the C4 genes, which included 2 bp (+TC) insertions at codon 1213 in exon 29, 1 bp deletions (-C) at codon 811 in exon 20, 1 bp (-C), 2 bp (-GT) deletions at codons 522 and 497 in exon 13 and null alleles. No mutations located at exons 13, 20 and 29 of the C4 gene, were detected amongst the patient and control samples in this study. C4A*Q0 was found in two out of the 130 control samples, while C4B*Q0 was present in two out of the 130 SLE patients. Overall, our results do not demonstrate a significant association to these known C4 mutations identified by previous studies, in the Malaysian scenario.