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Background and Aims: Fibroblast growth factor 21 (FGF21), an emerging metabolic hepatokine, is associated with atherosclerosis. An interaction with sex has been described in various populations. We aimed to study whether sex modulates the relationship between FGF21 and subclinical carotid atherosclerosis in a diabetes-enriched multiethnic population of Singapore. We explore differences in intermediary mechanisms, in terms of hypertension, lipids, and inflammation, between FGF21 and atherosclerosis.Methods: We recruited 425 individuals from a single diabetes center in Singapore, and demographics, anthropometry, metabolic profile, FGF21, and carotid ultrasonography were performed. Multivariable logistic regression models were used to study the association between subclinical atherosclerosis and FGF21 adjusting for age, ethnicity, body mass index (BMI), hemoglobin A1c (HbA1c), systolic and diastolic blood pressures, and low-density lipoprotein (LDL)-cholesterol separately for males and females as two groups after an interaction test.Results: An interaction test assessing interaction by sex on the relationship between subclinical atherosclerosis and FGF21 showed a significant interaction with sex (Pinteraction = 0.033). In the female subgroup, significant independent associations of standardized lnFGF21 with subclinical atherosclerosis were seen, with 1 SD increment in lnFGF21 being associated with 1.48-fold (95% CI: 1.03, 2.12; p = 0.036) increase in risk. In the male subgroup, the association of subclinical atherosclerosis with standardized lnFGF21 was not significant [odds ratio (OR) (95% CI): 0.90 (0.63, 1.28); p = 0.553]. We found sex interactions with pulse pressure being significantly associated in females only and triglycerides and C-reactive protein being associated with males only.Conclusion: FGF21 is positively associated with subclinical carotid atherosclerosis in women, but not in men. The sex–racial patterns in the mechanisms by which FGF21 causes subclinical atherosclerosis needs to be explored in larger population-based studies and mechanistically studied in greater detail.

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)(1 center dot 0)-MR5 center dot 0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4 center dot 0. Detection rates for deep-response samples were 95 center dot 7% at MR4 center dot 5, 78 center dot 3% at MR4 center dot 7 and 87 center dot 0% at MR5 center dot 0. Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29 center dot 3% to 69 center dot 0%. Linearity ranged from 0 center dot 9330 to 1 center dot 000 (average 0 center dot 9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.

BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).

Studies have shown that the genetic risk factors for rheumatoid arthritis (RA) differ substantially between Asian and Caucasian populations. Even among Asian populations, the genetic contributions of NLRP1, PTPN22 and PADI4 have been controversial. Consequently, we sought to address these separate findings and determine whether any of these proposed risk variants are associated with RA susceptibility, onset, DAS activity and erosion in a Singaporean Chinese cohort. We genotyped five SNPs within NLRP1 (rs878329 and rs6502867), PTPN22 (rs2488457 and rs6665194), and PADI4 (rs2240340) in 500 anti-cyclic citrullinated peptide antibody-positive (ACPA) patients with RA and 500 healthy controls using TaqMan assays. The CC genotype of NLRP1 rs878329 and TT genotype of PADI4 rs2240340 were associated with RA susceptibility. The risk association of the T allele of PADI4 rs2240340 with RA was confirmed through a meta-analysis based on previous reports in Asian populations. The GG genotype of PTPN22 rs6665194 (−3508A>G) was associated with significantly reduced risk of RA. No significant association was found for NLRP1 rs6502867 T/C and PTPN22 rs2488457 G/C polymorphisms. None of the five SNPs was associated with RA’s clinical features. This work supports the association of the T allele of PADI4 rs2240340 with RA in Asians. The roles of NLRP1 rs878329 G/C and PTPN22 rs6665194 A/G polymorphisms were demonstrated for the first time. We also propose rs6665194 to be a promising candidate for RA risk evaluation between ethnicities.

WHAT IS KNOWN AND OBJECTIVE Several Caucasian cohort studies have associated at least one loss-of-function CYP2C19 on Clopidogrel (LoF-Clopidogrel) with major adverse cardiovascular events (MACE), and only a couple have used Clinical Pharmacogenetics Implementation Consortium (CPIC® ) phenotype grouping to study the associations. We primarily aimed to study the impact of use of platelet reactivity testing to escalate antiplatelet therapy and secondarily to study the association of CPIC phenotype with MACE outcomes in South-East Asian Acute Coronary Syndrome (ACS) subjects. METHOD A retrospective genotype study was performed on 238 percutaneous coronary intervention subjects, originally planned for escalation of antiplatelets using platelet reactivity testing. RESULTS AND DISCUSSION There was no difference in MACE between the switched and unswitched groups; however, 'all bleeds' and 'clinically significant bleeds' (CSB) were statistically higher in the patients who were switched to prasugrel. The subgroup of patients who remained on clopidogrel (n = 199) were analysed using phenotype categories and MACE. Eleven percent (11.4%) of CYP2C19 poor metabolizers (PM) suffered MACE, compared with 1.3% of extensive metabolizers (EM). LoF-Clopidogrel patients are significantly more likely to experience MACE compared with non-LoF subjects (8.0% vs 5.4%, P: .041). WHAT IS NEW AND CONCLUSION In our multivariate analysis, LoF-Clopidogrel, malay ethnicity, diabetics and use of proton pump inhibitors were independent predictors of MACE. There were numerically more bleeds in LoF subjects who were on prasugrel compared with Clopidogrel (23.5% vs 11%, P = .082). Our data corroborate with current findings on platelet reactivity testing, suggesting that the assay would not be sensitive enough to pick up sufficient 'at-risk' subjects as compared to the use of CYP2C19 genotyping.

The haptoglobin 2-2 genotype is associated with atherosclerosis in type 2 diabetes mellitus. We examined the associations of the haptoglobin 2-2 genotype with C-reactive protein (high-sensitivity C-reactive protein) and carotid artery intima-media thickness, adjusting for age, gender, ethnicity, type 2 diabetes mellitus, smoking status, body mass index, blood pressure, glycated haemoglobin, non-high-density lipoprotein cholesterol and medications via logistic multivariate regression in 200 subjects (160 type 2 diabetes mellitus versus 40 healthy individuals). The prevalence of the haptoglobin 2-2 genotype was 58% (115/200), higher in the Indians than in Chinese (72% versus 45%, p = 0.001). Multivariate analysis showed that the haptoglobin 2-2 genotype was associated with high-sensitivity C-reactive protein [mean: 3.5 ± 3.9 versus 2.2 ± 2.6 mg/L (non-haptoglobin 2-2), p

Introduction There is limited diversity in current hereditary multi-gene testing data-sets, leading to greater challenges in accurate variant classification. To address this, a collaboration between 3 Singapore-based healthcare organizations (Tan Tock Seng Hospital, National Cancer Centre Singapore and Lucence Diagnostics) pooled results from patients of East Asian, Southeast Asian and South Asian ancestry, representing over 60% of the world's population. We hypothesized that a multi-ethnic collaboration would provide deeper understanding of cancer predisposition genes, particularly in terms of novel variants. Methods A total of 704 cancer patients of multi-ethnic Asian (SE Asian, East Asian and South Asian) ethnicity with either a history of breast, ovarian, pancreatic or prostate cancer, were tested with multi-gene panels. All patients were tested by multi-gene testing, including but not limited to BRCA1/2, PALB2 and ATM. In addition to genetic testing, the family histories of the patients were collected. All variants were classified by ACMG criteria. Chi-square testing was used for statistical analysis. Results Three sites (TTSH, NCCS, and Lucence) pooled patients selected for breast (n=458), ovarian (n=176), pancreatic (n=61) and prostate cancer (n=25). The mean and median ages were 44.9 years and 43 years, respectively. Of the 704 patients, 209 had a history of cancer in their first degree relatives, 432 did not and 63 patients did not know of any cancers in their first degree relatives. 122 of 704 patients (17.33%) had pathogenic/likely pathogenic variants in any tested risk-related gene, of which 86 were in BRCA1/2, 11 in PALB2 and 4 in ATM. Some variants were detected in more than one patient. 212 of 704 patients (30.1%) had VUSs detected in any tested risk-related gene, with 32.1% in BRCA1/2. There was a positive association between multiple-cancer status and pathogenic variants (9/22 vs 113/682, p = 0.007). Most notably, among the unique pathogenic/likely pathogenic variants in BRCA1, BRCA2, PALB2 and ATM, 10.64% (5/47), 12.9% (4/31), 30% (3/10) and 25% (1/4) respectively were novel variants, not previously reported in ClinVar (Dec 2019). Conclusion To our knowledge, this is the largest regional multi-ethnic cohort of patients with breast, ovarian, pancreatic and prostate cancer undergoing comprehensive genetic testing. Only one third of patients reported a first-degree family history suggesting that testing ought to be performed if clinical suspicion is high. Notably, 14.1% of BRCA1/BRCA2/PALB2/ATM pathogenic/likely pathogenic variants detected in our cohort were novel variants, not hitherto published in ClinVar. In conclusion, this collaboration demonstrates that testing of Asian patients can enrich global understanding of cancer predisposition gene mutations. This will improve cancer prevention, surveillance, and treatment selection for cancer patients, such as the use of PARP inhibitors for genetic defects of DNA repair. Citation Format: Jens Samol, Wei Lim Chia, Liuh Ling Goh, Matthew Myint, Min-Han Tan, Ru Jin Tay, Hao Chen, Yukti Choudhury, Ann SG Lee. Germline homologous recombination deficiency pathway defects in a multi-ethnic East Asian, Southeast Asian and South Asian cancer patient cohort [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4600.

Objectives: To study associations of established cardiovascular risk factors, inflammation and carotid artery intima-media thickness (CIMT) with oxidative stress in type 2 diabetes mellitus (T2DM). Methods: We recruited 343 T2DM subjects from the three main ethnic groups in Singapore (Chinese: 196, Indians: 106, Malays: 41; Age: 54.7±10.4 years; Males: 46%). Measurements: Demographics, BMI, blood pressure, HbA1c, glucose and lipids; CIMT and C-reactive protein (hsCRP). Derivatives of reactive oxygen metabolites (dROMS) and total anti-oxidant status (TAC) was estimated by the automated clinical chemistry analyser (Diacron, Italy). The total oxidative index (OXY) was calculated as the difference between standardised dROMS and TAC values. The relationship between OXY, dROMS and TAC as dependent variable and age, gender, ethnicity, BMI, blood pressure, HbA1c, fasting lipids, log CRP and CIMT as co-variates was assessed using multiple regression analysis. Associations with gender and ethnicity were assessed by student’s t test and Kruskal Wallis test respectively. Results: The mean OXY was higher in the females (0.2±1.3) compared to the males (0.2±1.0); p Conclusions: In T2DM, total oxidative stress is a function of HbA1c, non-HDL-Cholesterol and low-grade inflammation. HbA1c and inflammation increase reactive oxygen metabolites, whereas non-HDL cholesterol decreases the anti-oxidant potential. The mechanisms of oxidative stress generation, in particular in various ethnic groups, needs to be studied further. Disclosure R. Dalan: None. L. Goh: None. X. Tang: None. D.E.K. Chew: None. B. Boehm: None.

We read the recent studies by Lee et al1 and the IGNITE network (Implementing Genomics in Practice) with much interest and wish to share findings from our cohort of 247 Asians in Khoo Teck Puat Hospital, Singapore. To our knowledge, we are the first Asian group to demonstrate similar interventional cohort findings as those of Lee et al1 and Cavallari et al from the IGNITE network.2 We conducted a single-center, prospective, open label study in 247 Asian patients who underwent percutaneous coronary intervention and received dual antiplatelet therapy consisting of aspirin and clopidogrel. At the time of design, we intended to switch subjects with high-on-treatment response from clopidogrel to prasugrel. Additional blood samples were collected for genotyping. High-on-treatment …

Objective To evaluate the feasibility of detecting actionable gene mutations in circulating tumor DNA (ctDNA) in patients with advanced non-small-cell lung cancer (NSCLC) using targeted next-generation sequencing (NGS). Materials and methods In total 50 plasma samples from patients newly diagnosed with advanced NSCLC or resistant to first-line tyrosine kinase inhibitors (TKIs) were subjected to deep sequencing on a seven-gene panel (BRAF, EGFR, ERBB2, KRAS, NRAS, PIK3CA, PTEN) incorporated with molecular barcodes to improve accuracy in variant detection. When possible, results were compared with those from matched tissue samples. Results At least one alteration in the ctDNA was detected in 44 out of 50 patients (88%); EGFR was the most frequently mutated gene. Half the total number of patients (50%, 25 of 50) had at least one actionable genetic alteration with targeted therapies available for treatment. Our results showed a high concordance rate of 81% in detection of EGFR mutation between 26 matched tissue and plasma samples. For progressive patients, from whom tissue is mostly unavailable, the resistant EGFR T790 M mutation was validated using the droplet digital polymerase chain reaction (ddPCR), yielding a concordance of 92% between alternative platforms. Conclusion Our study demonstrated that therapeutically actionable mutations can be detected with high accuracy in ctDNA using NGS. This promising approach offers alternative and non-invasive diagnostic methods for treatment guidance and clinical monitoring.

Background Lipid storage myopathy (LSM) is a diverse group of lipid metabolic disorders with great variations in the clinical phenotype and age of onset. Classical multiple acyl-CoA dehydrogenase deficiency (MADD) is known to occur secondary to mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene. Whole exome sequencing (WES) with clinical correlations can be useful in identifying genomic alterations for targeted therapy. Case presentation We report a patient presented with severe muscle weakness and exercise intolerance, suggestive of LSM. Diagnostic testing demonstrated lipid accumulation in muscle fibres and elevated plasma acyl carnitine levels. Exome sequencing of the proband and two of his unaffected siblings revealed compound heterozygous mutations, c.250G > A (p.Ala84Thr) and c.770A > G (p.Tyr257Cys) in the ETFDH gene as the probable causative mutations. In addition, a previously unreported variant c.1042C > T (p.Arg348Trp) in ACOT11 gene was found. This missense variant was predicted to be deleterious but its association with lipid storage in muscle is unclear. The diagnosis of MADD was established and the patient was treated with riboflavin which resulted in rapid clinical and biochemical improvement. Conclusions Our findings support the role of WES as an effective tool in the diagnosis of highly heterogeneous disease and this has important implications in the therapeutic strategy of LSM treatment. Electronic supplementary material The online version of this article (10.1186/s12920-018-0356-8) contains supplementary material, which is available to authorized users.

Objective: The haptoglobin 2-2 genotype is associated with lower haptoglobin concentrations and atherosclerosis in diabetes. Endothelial cell apoptosis contributes significantly to atherosclerosis. We studied endothelial cell apoptosis in diabetes patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype. Approach and results: We pooled plasma from 10 patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype and quantified endothelial cell apoptosis using a hemodynamic lab-on-chip system. Then, we conducted similar experiments on individual diabetes plasma samples with the haptoglobin 2-2 (n = 20) and non-haptoglobin 2-2 genotype (n = 20). Haptoglobin beta concentrations were measured by Western blot analysis. We looked for association with demographic, metabolic variables, inflammation and oxidative stress. In pooled plasma, endothelial cell apoptosis was higher in haptoglobin 2-2 group (haptoglobin 2-2: 23.18% vs non-haptoglobin 2-2:15.32%). In individual samples, univariate analysis showed that endothelial cell apoptosis correlated with haptoglobin beta concentration [β = −10.29 (95% confidence interval: −13.44, −7.14), p 0.05). Conclusion: These results show that regardless of the haptoglobin genotype, haptoglobin is associated with prevention of endothelial cell apoptosis in diabetes.

Background Genetic determinants of drug response remain stable throughout life and offer great promise to patient-tailored drug therapy. The adoption of pharmacogenetic (PGx) testing in patient care requires accurate, cost effective and rapid genotyping with clear guidance on the use of the results. Hence, we evaluated a 32 SNPs panel for implementing PGx testing in clinical laboratories. Methods We designed a 32-SNP panel for PGx testing in clinical laboratories. The variants were selected using the clinical annotations of the Pharmacogenomics Knowledgebase (PharmGKB) and include polymorphisms of CYP2C9, CYP2C19, CYP2D6, CYP3A5 and VKORC1 genes. The CYP2D6 gene allele quantification was determined simultaneously with TaqMan copy number assays targeting intron 2 and exon 9 regions. The genotyping results showed high call rate accuracy according to concordance with genotypes identified by independent analyses on Sequenome massarray and droplet digital PCR. Furthermore, 506 genomic samples across three major ethnic groups of Singapore (Malay, Indian and Chinese) were analysed on our workflow. Results We found that 98% of our study subjects carry one or more CPIC actionable variants. The major alleles detected include CYP2C9*3, CYP2C19*2, CYP2D6*10, CYP2D6*36, CYP2D6*41, CYP3A5*3 and VKORC1*2. These translate into a high percentage of intermediate (IM) and poor metabolizer (PM) phenotypes for these genes in our population. Conclusion Genotyping may be useful to identify patients who are prone to drug toxicity with standard doses of drug therapy in our population. The simplicity and robustness of this PGx panel is highly suitable for use in a clinical laboratory.

Underrepresentation of Asian genomes has hindered population and medical genetics research on Asians, leading to population disparities in precision medicine. By whole-genome sequencing of 4,810 Singapore Chinese, Malays, and Indians, we found 98.3 million SNPs and small insertions or deletions, over half of which are novel. Population structure analysis demonstrated great representation of Asian genetic diversity by three ethnicities in Singapore and revealed a Malay-related novel ancestry component. Furthermore, demographic inference suggested that Malays split from Chinese ∼24,800 years ago and experienced significant admixture with East Asians ∼1,700 years ago, coinciding with the Austronesian expansion. Additionally, we identified 20 candidate loci for natural selection, 14 of which harbored robust associations with complex traits and diseases. Finally, we show that our data can substantially improve genotype imputation in diverse Asian and Oceanian populations. These results highlight the value of our data as a resource to empower human genetics discovery across broad geographic regions. Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC) National Research Foundation (NRF) Accepted version We acknowledge H.M. Kang, S. Das, A. Tan, F. Zhang, J. Terhorst, P.-R. Loh, and G. Hellenthal for helpful discussions and support from all participants and clinical research coordinators of the contributing cohorts and studies: the TTSH Healthy Control Workgroup, the SEED cohort, the Asian Sudden Cardiac Death in Heart Failure Study, the Singapore Heart Failure Outcomes and Phenotypes (SHOP) cohort, the Asian neTwork for Translational Research and Cardiovascular Trials (ATTRaCT), the Parkinson’s Disease Study, the Peranakan Genome Study, the Platinum Asian Genomes Project, the Bariatric Surgery Study, the National Heart Centre Singapore Biobank and SingHEART cohorts, and the GUSTO and S-PRESTO study groups. This study was supported by Singapore’s A*STAR (core funding and IAF-PP H17/01/a0/007), BMRC (SPF2014/001, SPF2013/002, SPF2014/003, SPF2014/004, and SPF2014/005), NMRC (CIRG/1371/2013, CIRG/1417/2015, CIRG/1488/ 2018, CSA-SI/0012/2017, CG/017/2013, CG/M006/2017_NHCS, TCR/013- NNI/2014, STaR/0011/2012, STaR2013/001, STaR/014/2013, STaR/0026/ 2015, TCR/006-NUHS/2013, TCR/012-NUHS/2014, TCR/004-NUS/2008, TCR/012-NUHS/2014, and center grants 2010-13 and 2013-2017), NRF (NRFF2016-03), National University of Singapore, SingHealth and DukeNUS, and Alexandra Health small innovative grant SIGII/15203 and funding from Huazhong University of Science and Technology, the Tanoto Foundation, the Lee Foundation, the Boston Scientific Investigator Sponsored Research Program and Bayer, the NSF (DEB-1753489), and the Alfred P. Sloan Foundation. The computation was partially performed on resources of the National Supercomputing Centre, Singapore (https://www.nscc.sg).

Falcipains (FP) of Plasmodium falciparum are important virulence factors marked as potential targets for antimalarial drug discovery. In this study, the previously uncharacterized fp2B (PF11_0161) was shown to be highly expressed as an active enzyme during the erythrocytic stage. With three related proteases in the FP family and the existence of human homologues, it is prudent to identify clusters of residues unique to the parasite proteases that can be targeted selectively for drug design. Using bioinformatic tools, we have carefully mapped out a highly conserved and unique region constituted by I85, S149, and A151 in the plasmodial proteases that can influence the development of compounds capable of inhibiting the entire FP family. Taking drug interactions with the human homologues into consideration, these residues in FP2B were replaced with the cognate residues found in human cathepsin L (catL) for evaluation. Despite the high sequence similarity between the FP2 isozymes (97.5%), FP2B is found to be more tolerant to amino acid substitution at position 149 than FP2A. This structural disparity implied that residues mediating peptide substrate interactions are not fully conserved across the FP family and warrant attention in the design and evaluation of protease inhibitors focused on the FPs. The simultaneous substitution of the neighboring residues (I85 or A151) rendered the double mutants (S149A/I85M and S149A/A151D) completely inactive. Significantly, the mutations did not result in ‘catL-like’ specificity, suggesting that substrate-based inhibitors could be rationally designed against these important parasite-specific structural determinants.

Falcipains form a class of papain-like cysteine proteases found in Plasmodium falciparum. This group of proteases has been suggested to be promising targets for anti-malarial chemotherapy. Despite being the first falcipain to be identified, the physiological role(s) of falcipain 1 (fp1) remains a mystery. Its suggested functions include haemoglobin degradation, erythrocytic invasion and oocyst production. In this study, the procurement of the gene coding for fp1 and its soluble expression in a heterologous host, Escherichia coli, have enabled further enzyme characterization. The recombinant fp1 protease was found to be unlike falcipain 2 (fp2A) in being more active at neutral pH than at acidic pH against the Z-LR-AMC fluorogenic substrate, suggesting a probable localization in the cytosol and not in the food vacuole. Interestingly, a common cysteine specific inhibitor, E64, did not inhibit fp1 activity, indicating dissimilar biochemical characteristics of fp1 from the other falcipains. This may be explained by computational analysis of the primary structures of the falcipain isozymes, as well as that of papain. The analysis revealed that Tyr61 (papain numbering), which is correspondingly absent in fp1, might be an important residue involved in E64 substrate binding.

The hemoglobin-degrading cysteine proteases falcipains of the malaria parasite Plasmodium falciparum are regarded as potential drug targets. Despite their obvious importance in the virulence of malaria, these proteases remain poorly characterized at the structural levels. Using a bioinformatic and site-directed mutagenesis approach, residues essential for the structure and function of FP2A are elucidated in this study. In total, nine mutants of FP2A were constructed to test the proposed importance of seven different amino acid residues. These recombinant protease mutants were solubly expressed in Escherichia coli and purified by affinity chromatography for enzymatic assessments. Notably, substitutions at positions C99 and C119 induce structural alterations and led to significant reduction in enzyme activity (>97%). The analyses also validated the role of the active triad comprising of C42, H174, and N204 in catalysis and identified a serine at position 149 which is required for specific peptide substrate interactions. The parasite-specific residues, C99, C119, and S149, represent potential sites for differential targeting, since the corresponding residues are absent in the human host's isozymes.

Falcipain-2 (fp2) is a hemoglobinase required for supplying peptides and amino acids for the proliferation of Plasmodium falciparum in blood. The prospect of circumventing its activity thereby serves as a potential strategy for mining drugs for anti-malarial therapy. However, to date, efforts to express soluble and active fp2 in Escherichia coli have been futile. To overcome this problem, fp2 was expressed under an array of conditions including the exploitation of multiple gene constructs in eukaryotic and prokaryotic hosts. A series of experiments led to the finding that the placement of maltose-binding protein (MBP) before the fp2 mature domain was best in availing the soluble expression of the protease. The results also indicate that the prodomain impaired the bacterial expression of the protease and the amino acid residues at the N-terminal segment of mature fp2 can have a significant effect on the folding and solubility of the enzyme. The overexpressed MBP-fp2 fusion protein was purified and shown to be functionally active, providing a very useful alternative to the use of resolubilized enzyme for future study of structure and function of fp2.

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.