Genetic Risk Factors of Systemic Lupus Erythematosus in the Malaysian Population: A Minireview

SLE is an autoimmune disease that is not uncommon in Malaysia. In contrast to Malays and Indians, the Chinese seem to be most affected. SLE is characterized by deficiency of body's immune response that leads to production of autoantibodies and failure of immune complex clearance. This minireview attempts to summarize the association of several candidate genes with risk for SLE in the Malaysian population and discuss the genetic heterogeneity that exists locally in Asians and in comparison with SLE in Caucasians. Several groups of researchers have been actively investigating genes that are associated with SLE susceptibility in the Malaysian population by screening possible reported candidate genes across the SLE patients and healthy controls. These candidate genes include MHC genes and genes encoding complement components, TNF, FcγR, T-cell receptors, and interleukins. However, most of the polymorphisms investigated in these genes did not show significant associations with susceptibility to SLE in the Malaysian scenario, except for those occurring in MHC genes and genes coding for TNF-α, IL-1β, IL-1RN, and IL-6

Development of insulated isothermal PCR for rapid on-site malaria detection

Background Detection of Plasmodium spp. is sometimes inconvenient especially in rural areas that are distant from a laboratory. In this study a portable diagnostic test of Plasmodium spp. was developed using insulated isothermal polymerase chain reaction (iiPCR) as an alternative approach to improve this situation. Methods A pair of universal primers and probe were designed to amplify and detect gene encoding 18S small sub-unit rRNA of Plasmodium spp using iiPCR method in a portable device, POCKIT™. The efficiency and detection limit of the assay were evaluated using quantitative real-time polymerase chain reaction (qPCR) approach before being subjected to testing in POCKIT™. Detection results of POCKIT™ were displayed as ‘+’, ‘−’ or ‘?’ based on the fluorescence ratio after/before reaction. A total of 55 and 35 samples from malaria patients and healthy subjects, respectively, were screened to evaluate the feasibility of this newly designed iiPCR assay. Results The iiPCR assay allowed the detection of various species of Plasmodium, including those infecting humans (Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale), monkeys, birds, and rodents. Efficiency of the assay achieved 96.9 % while the lower detection limit was ≥100 copies of plasmodial DNA. Specificity of the assay was assured as it could not detect human, bacterial and other parasitic DNA. Among the 55 clinical samples tested, 47 (85.4 %) of them were detected as positive by POCKIT™. Four (7.3 %) samples with fluorescence ratio after/before reaction of <1.2 were reported as negative while another four (7.3 %) were ambiguously detected as they had fluorescence ratios between 1.2 and 1.3. The fluorescence ratio was not found to be associated with the copy number of plasmodial DNA. This approach can only be considered as a qualitative method. Conclusions The portable iiPCR system may serve as an alternative approach for preliminary screening of malaria in endemic rural areas. The system may also be useful for detecting animal malaria in the field. Although it is not as quantitative as qPCR method, it is comparatively fast and easy to handle. It is believed that the POCKIT-iiPCR assay is able to achieve 100 % sensitivity if increased amount of DNA from each sample is used. The iiPCR assay can also be upgraded in future to detect multiple Plasmodium spp. at the same time by designing the specific primers and probes

Detection and determination of furfural in crude palm oil.

In the palm oil mill, fresh fruit bunch (FFB) undergoes various thermal and mechanical treatments to produce the crude palm oil (CPO). FFB consists of many fruits attached to the spikelets that are spirally arranged on the main bunch stalk. Each fruit is made up of a nut enveloped by the fleshy mesocarp, which is reinforced by strands of fibers running from the base towards the fruit tip. A ripe fruit mesocarp contains oil-rich cellulosic cells. These cells are bound together by hemicellulose. Whilst cellulose is very stable, the hemicellulose is easily hydrolyzed. This hydrolysis occurs during sterilization of the FFB when it is exposed to temperatures of 140-145°C and pressure of 40-45 pound per square inch (psi) or 275.8-310.3 kPa for 1-1½ hours. This condition aims at and ensures the detachment of fruits from the bunch. The in-depth chemical changes that occur in the FFB during sterilization are not fully understood and continuously being investigated. Xyloses form one of the products of hydrolysis, and furfural is another product that results from the dehydration of pentose formed also upon the hydrolysis of hemicellulose. Presence of furfural was tested in six extracted samples, namely CPO, mill-pressed crude, condensate oil, sludge oil, sterilized FFB oil and unsterilized FFB oil, using aniline acetate colorimetric method, thin-layer chromatography (TLC) and UV-visible spectrophotometry. The color formation was compared to that of standard furfural. Furfural was detected in CPO, crude, condensate oil, sludge oil and sterilized FFB oil, while it was undetected in the unsterilized FFB. The amount of furfural was quantified in CPO, condensate oil and sludge oil using high-performance liquid chromatography (HPLC)

Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device

Background. Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. Method. In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC nontargeted strains were tested with iiPCR and qPCR in parallel. Results. All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45– 99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. Conclusion. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen

Development of high resolution melting analysis for the diagnosis of human malaria

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis

Genetic diversity of circumsporozoite protein in Plasmodium knowlesi isolates from Malaysian Borneo and Peninsular Malaysia

Understanding the genetic diversity of candidate genes for malaria vaccines such as circumsporozoite protein (csp) may enhance the development of vaccines for treating Plasmodium knowlesi. Hence, the aim of this study is to investigate the genetic diversity of non-repeat regions of csp in P. knowlesi from Malaysian Borneo and Peninsular Malaysia. A total of 46 csp genes were subjected to polymerase chain reaction amplification. The genes were obtained from P. knowlesi isolates collected from different divisions of Sabah, Malaysian Borneo, and Peninsular Malaysia. The targeted gene fragments were cloned into a commercial vector and sequenced, and a phylogenetic tree was constructed while incorporating 168 csp sequences retrieved from the GenBank database. The genetic diversity and natural evolution of the csp sequences were analysed using MEGA6 and DnaSP ver. 5.10.01. A genealogical network of the csp haplotypes was generated using NETWORK ver. 4.6.1.3. The phylogenetic analysis revealed indistinguishable clusters of P. knowlesi isolates across different geographic regions, including Malaysian Borneo and Peninsular Malaysia. Nucleotide analysis showed that the csp nonrepeat regions of zoonotic P. knowlesi isolates obtained in this study underwent purifying selection with population expansion, which was supported by extensive haplotype sharing observed between humans and macaques. Novel variations were observed in the C-terminal non-repeat region of csp. The csp non-repeat regions are relatively conserved and there is no distinct cluster of P. knowlesi isolates from Malaysian Borneo and Peninsular Malaysia. Distinctive variation data obtained in the C-terminal non-repeat region of csp could be beneficial for the design and development of vaccines to treat P. knowlesi

Development of insulated isothermal PCR for rapid on-site malaria detection

Background Detection of Plasmodium spp. is sometimes inconvenient especially in rural areas that are distant from a laboratory. In this study a portable diagnostic test of Plasmodium spp. was developed using insulated isothermal polymerase chain reaction (iiPCR) as an alternative approach to improve this situation. Methods A pair of universal primers and probe were designed to amplify and detect gene encoding 18S small sub-unit rRNA of Plasmodium spp using iiPCR method in a portable device, POCKIT™. The efficiency and detection limit of the assay were evaluated using quantitative real-time polymerase chain reaction (qPCR) approach before being subjected to testing in POCKIT™. Detection results of POCKIT™ were displayed as ‘+’, ‘−’ or ‘?’ based on the fluorescence ratio after/before reaction. A total of 55 and 35 samples from malaria patients and healthy subjects, respectively, were screened to evaluate the feasibility of this newly designed iiPCR assay. Results The iiPCR assay allowed the detection of various species of Plasmodium, including those infecting humans (Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale), monkeys, birds, and rodents. Efficiency of the assay achieved 96.9 % while the lower detection limit was ≥100 copies of plasmodial DNA. Specificity of the assay was assured as it could not detect human, bacterial and other parasitic DNA. Among the 55 clinical samples tested, 47 (85.4 %) of them were detected as positive by POCKIT™. Four (7.3 %) samples with fluorescence ratio after/before reaction of <1.2 were reported as negative while another four (7.3 %) were ambiguously detected as they had fluorescence ratios between 1.2 and 1.3. The fluorescence ratio was not found to be associated with the copy number of plasmodial DNA. This approach can only be considered as a qualitative method. Conclusions The portable iiPCR system may serve as an alternative approach for preliminary screening of malaria in endemic rural areas. The system may also be useful for detecting animal malaria in the field. Although it is not as quantitative as qPCR method, it is comparatively fast and easy to handle. It is believed that the POCKIT-iiPCR assay is able to achieve 100 % sensitivity if increased amount of DNA from each sample is used. The iiPCR assay can also be upgraded in future to detect multiple Plasmodium spp. at the same time by designing the specific primers and probes

Molecular pathology of systemic lupus erythematosus in Asians

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease affecting various parts of the body. Polymorphisms in genes involved in toll-like receptor (TLR)/interferon (IFN) signalling pathways have been reported previously to be associated with SLE in many populations. This study aimed to investigate the role of seven single nucleotide polymorphisms (SNPs) within TNFAIP3 (rs2230936 and rs3757173), STAT4 (rs7574865, rs10168266, and rs7601754), and IRF5 (rs4728142 and rs729302), that are involved in upstream and downstream pathway of type I IFN production, in the Malaysian SLE. Genotyping of 360 Malaysian SLE patients and 430 normal healthy individuals revealed that STAT4 rs7574865 and rs10168266 with their minor T alleles [p=0.001, odds ratio (OR)=1.40 and p=5.75x10-4, OR=1.43, respectively] and TNFAIP3 rs3757173 with its C allele (p=0.017, OR=1.66) were associated with elevated risk of SLE in the Malaysian patients, as well as in the Malays and Chinese. The minor G allele of TNFAIP3 rs2230926 was found to reduce the SLE risk (p=0.021, OR=0.53) in the Malaysian patients, particularly in the Malays. No association was observed for STAT4 rs7601754 and SNPs in IRF5 gene. Besides having haplotype TT (p=1 x 10-4, OR=1.53) and CG (p=0.02, OR=0.76) being significantly associated with SLE susceptibility, STAT4 rs7574865 and rs10168266 also formed the best model for high-risk group in multifactor dimensionality reduction (MDR) test. In conclusion, polymorphisms in STAT4 and TNFAIP3 genes could be potential genetic risk factors for SLE development in the Malaysian individuals. Genotyping analysis of two recently reported HLA variants demonstrated that the frequency for minor allele G and its homozygous GG of HLA-DRB1/HLA-DQA1 rs9271366 significantly higher in the Malaysian SLE patients (p=5.78 x 10-5, OR=1.63 and p=0.001, OR=3.30, respectively), as well as in the Malays and Chinese. Whereas, the minor allele T (p=1.93 x 10-5, OR=0.58) and the heterozygous CT (p=3.65 x 10-4, OR=0.54) of HLA-DQB1/HLA-DQA2 rs9275328 conferred protection to SLE in the Malaysians, including the Malays and Chinese. Both SNPs did not show associations with SLE in the Indians. Haplotype GC and AT were significantly associated with SLE (p<5.0 x 10-4) after 10000 permutations. The MDR test clustered the genotype combinations of GG and CC, and AG and CC of rs9271366 and rs9275328, accordingly, as high-risk group. These results consolidated the importance of HLA gene, which involved in the immune complex processing, in the Malaysian SLE. The seeking of potential SLE/lupus nephritis (LN)-associated protein biomarkers was attempted using two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches. A total of 30 spots from 18 high-abundant (HAP)-depleted plasma proteins, 26 spots from 11 plasma HAPs, and 59 spots from 25 urinary proteins were identified to have differential expressions. Serum constitutional and transport proteins, including haptoglobin and histidine-rich glycoprotein, occupied the largest portion among both plasma and urinary proteins (~28%). Several proteins were newly discovered in the present study, such as afamin, hemopexin, retinol-binding protein 4, and vitamin D-binding protein. Group-specific proteins were also determined for each group of SLE/LN, for instance up-regulation of plasma alpha-1-antitrypsin and down-regulation of serum amyloid P-component were unique to SLE without kidney involvement, while decreased urinary protein AMBP was specific to LN class V. These proteins may have prospective diagnostic value to discriminate the types of SLE and classes of LN. Most differentially expressed plasma proteins returned to normal levels upon LN remission, while most urinary proteins remained at their abnormal levels. Interactions between SLE/LN-associated proteins analysed using IPA software summarized the networks or pathways responsible for the pathogenesis of each patient group. Network functions in cellular movement, inflammatory response, and cancer was associated with SLE without kidney involvement, whereas the top scored network generated for LN class II involved in cellular movement, haematological system development and function, and immune cell trafficking. Abnormal lipid metabolism, small molecule biochemistry, molecular transport, and vitamin and mineral metabolism were responsible for the development of LN class III. LN class IV was related to network implicated in cell cycle, cell death and survival, and tumor morphology. Connective tissue disorders, inflammatory disease, and skeletal and muscular disorders might increase the susceptibility to LN class V. Interestingly, networks having functions in immune and inflammatory responses were not as important as expected. Mycophenolate mofetil (MMF) is one of the promising immunosuppressants with less toxicity used in the treatment of LN. After 8 months of MMF treatment, LN class III patients showed level changes in 18 protein fragments derived from 10 plasma proteins, such as angiotensinogen, and 6 protein fragments from 4 urinary proteins, such as lysosomal alpha-glucosidase. The results might have uncovered the mechanism of action of MMF other than that has been described previously

Molecular pathology of systemic lupus erythematosus in Asians

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease affecting various parts of the body. Polymorphisms in genes involved in toll-like receptor (TLR)/interferon (IFN) signalling pathways have been reported previously to be associated with SLE in many populations. This study aimed to investigate the role of seven single nucleotide polymorphisms (SNPs) within TNFAIP3 (rs2230936 and rs3757173), STAT4 (rs7574865, rs10168266, and rs7601754), and IRF5 (rs4728142 and rs729302), that are involved in upstream and downstream pathway of type I IFN production, in the Malaysian SLE. Genotyping of 360 Malaysian SLE patients and 430 normal healthy individuals revealed that STAT4 rs7574865 and rs10168266 with their minor T alleles [p=0.001, odds ratio (OR)=1.40 and p=5.75x10-4, OR=1.43, respectively] and TNFAIP3 rs3757173 with its C allele (p=0.017, OR=1.66) were associated with elevated risk of SLE in the Malaysian patients, as well as in the Malays and Chinese. The minor G allele of TNFAIP3 rs2230926 was found to reduce the SLE risk (p=0.021, OR=0.53) in the Malaysian patients, particularly in the Malays. No association was observed for STAT4 rs7601754 and SNPs in IRF5 gene. Besides having haplotype TT (p=1 x 10-4, OR=1.53) and CG (p=0.02, OR=0.76) being significantly associated with SLE susceptibility, STAT4 rs7574865 and rs10168266 also formed the best model for high-risk group in multifactor dimensionality reduction (MDR) test. In conclusion, polymorphisms in STAT4 and TNFAIP3 genes could be potential genetic risk factors for SLE development in the Malaysian individuals. Genotyping analysis of two recently reported HLA variants demonstrated that the frequency for minor allele G and its homozygous GG of HLA-DRB1/HLA-DQA1 rs9271366 significantly higher in the Malaysian SLE patients (p=5.78 x 10-5, OR=1.63 and p=0.001, OR=3.30, respectively), as well as in the Malays and Chinese. Whereas, the minor allele T (p=1.93 x 10-5, OR=0.58) and the heterozygous CT (p=3.65 x 10-4, OR=0.54) of HLA-DQB1/HLA-DQA2 rs9275328 conferred protection to SLE in the Malaysians, including the Malays and Chinese. Both SNPs did not show associations with SLE in the Indians. Haplotype GC and AT were significantly associated with SLE (p<5.0 x 10-4) after 10000 permutations. The MDR test clustered the genotype combinations of GG and CC, and AG and CC of rs9271366 and rs9275328, accordingly, as high-risk group. These results consolidated the importance of HLA gene, which involved in the immune complex processing, in the Malaysian SLE. The seeking of potential SLE/lupus nephritis (LN)-associated protein biomarkers was attempted using two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches. A total of 30 spots from 18 high-abundant (HAP)-depleted plasma proteins, 26 spots from 11 plasma HAPs, and 59 spots from 25 urinary proteins were identified to have differential expressions. Serum constitutional and transport proteins, including haptoglobin and histidine-rich glycoprotein, occupied the largest portion among both plasma and urinary proteins (~28%). Several proteins were newly discovered in the present study, such as afamin, hemopexin, retinol-binding protein 4, and vitamin D-binding protein. Group-specific proteins were also determined for each group of SLE/LN, for instance up-regulation of plasma alpha-1-antitrypsin and down-regulation of serum amyloid P-component were unique to SLE without kidney involvement, while decreased urinary protein AMBP was specific to LN class V. These proteins may have prospective diagnostic value to discriminate the types of SLE and classes of LN. Most differentially expressed plasma proteins returned to normal levels upon LN remission, while most urinary proteins remained at their abnormal levels. Interactions between SLE/LN-associated proteins analysed using IPA software summarized the networks or pathways responsible for the pathogenesis of each patient group. Network functions in cellular movement, inflammatory response, and cancer was associated with SLE without kidney involvement, whereas the top scored network generated for LN class II involved in cellular movement, haematological system development and function, and immune cell trafficking. Abnormal lipid metabolism, small molecule biochemistry, molecular transport, and vitamin and mineral metabolism were responsible for the development of LN class III. LN class IV was related to network implicated in cell cycle, cell death and survival, and tumor morphology. Connective tissue disorders, inflammatory disease, and skeletal and muscular disorders might increase the susceptibility to LN class V. Interestingly, networks having functions in immune and inflammatory responses were not as important as expected. Mycophenolate mofetil (MMF) is one of the promising immunosuppressants with less toxicity used in the treatment of LN. After 8 months of MMF treatment, LN class III patients showed level changes in 18 protein fragments derived from 10 plasma proteins, such as angiotensinogen, and 6 protein fragments from 4 urinary proteins, such as lysosomal alpha-glucosidase. The results might have uncovered the mechanism of action of MMF other than that has been described previously