Development of a local warfarin dosage guideline based on pharmacogenomics and haemostatic markers

Warfarin, the mainstream oral anticoagulant, has a narrow therapeutic index and wide interindividual dose variability, rendering maintaining its optimal dose in each individual a difficult task. Many warfarin dosing models have been developed worldwide in order to improve the accuracy of currently used international normalised ratio (INR) dosing method. However, those dosing models were not practical to be used due to extensive additional data that are required for dose calculation. In this study, a simpler warfarin's dosing model for local population has been studied for warfarin therapy reinitiation based on clinical, laboratory and genetic data. A total of 130 of patients on warfarin treatment in Hospital Universiti Sains Malaysia were recruited for the model-building. Patients' clinical data were extracted from the hospital database. Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) methods were used for genotyping of CYP2C9*2, *3 and VKORC1 -1639G>A while a newly developed nested allele-specific multiplex PCR was used for genotyping of VKORC1 381, 861, 5808 and 9041. Genotype data of VKORC1 381, 861, 5808 and 9041 were used to infer VKORC1 haplotype. The activity of vitamin K-dependent (VKD) clotting factors II, VII, IX and X were measured by using a benchtop haemostatic analyser. A newly developed and validated high performance liquid chromatography (HPLC) method with UV detector was used for measurement of serum warfarin levels that were subsequently used for pharmacokinetics data calculation in 24 patients. The warfarin's dosing model was developed by using a forward multiple linear regression. The heterozygous mutant genotype of CYP2C9*2 and *3 were rare (both at 3.8%), while the homozygous mutant was not detected. The frequency of VKORC1 -1639G>A andVKORC1 381 were similar. The genotype with highest frequency was the low warfarin's dose requirement genotype (GG: 54.6%). The VKORC1 H1H1, H1H7 or H1H9 and H7H7 were the most common haplotype pairs (53.1, 32.5 and 10.0%). All VKD clotting factor activities were not significantly associated with warfarin's dose requirement or with the INR. Maximum serum concentration, half-life and clearance of warfarin were not significantly associated with any genetic data or warfarin's dose requirement. The final warfarin's dosing model consists of age, the number of VKORC1 381 allele, mean INR and history of mitral valve replacement as useful predictor factors. The predictor factors explained 45.6% of warfarin dose variability. The developed dosing model is suitable to be used as guideline to determine the warfarin dose of patients who need to reinitiate a warfarin therapy

A New Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for Haplotyping of VKORC1 Gene to Predict Warfarin Sensitivity

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 analysiswas developed and validated. ExtractedDNAwas 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

Determinants for Healthy Lifestyle of Patients with Familial Hypercholesterolaemia

Lifestyle modification is a pivotal intervention for Familial Hypercholesterolaemia (FH). This study aims to describe the lifestyles (physical activity and healthy diet) and their associations with sociodemography, illness characteristics, psychological elements, family support and level of barrier. 100 participants were given Pro forma questionnaires to assess sociodemography and illness characteristics. The lifestyles, psychological elements, family support and level of barrier were assessed using the Theory of Planned Behaviour questionnaire. The determinants of healthy lifestyles include the status of receiving treatment, level of barrier and intention for behavioural change. The findings may inform the strategy for lifestyle modification of FH patients.Keywords: Familial Hypercholesterolaemia; lifestyle; physical activity; healthy diet.eISSN: 2398-4287© 2020. The Authors. Published for AMER ABRA cE-Bs by e-International Publishing House, Ltd., UK. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of AMER (Association of Malaysian Environment-Behaviour Researchers), ABRA (Association of Behavioural Researchers on Asians) and cE-Bs (Centre for Environment-Behaviour Studies), Faculty of Architecture, Planning & Surveying, Universiti Teknologi MARA, Malaysia.DOI: https://doi.org/10.21834/ebpj.v5i14.233

Determining Predictors of Depression and Anxiety for Prevention of Common Mental Illness among Staff of an Academic Institution in Malaysia

The Adopt-A-Park Programme has Information on depression, anxiety and predictors for these mental illnesses among the staff of the academic institution is sparse. Hence, this study aimed to determine the prevalence of these mental illnesses and investigate possible predictors. Depression, Anxiety and Stress Scale 21-item and pro forma questionnaires were used to assess the presence of depression, anxiety, sociodemographic, personal and job-related factors. Of 278 participants, 27.7% had depression, and 26.7% had anxiety. Predictors for depression include inadequate workplace facilities, low-tier job category, working in urban campus and low income. Predictors for clinical anxiety were high workplace responsibility and low-tier job category. Keywords: Depression; Anxiety; Academic Institution; Staff 2398-4279 © 2019 The Authors. Published for AMER ABRA CE-Bs by E-International Publishing House, Ltd., UK. This is an open-access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of AMER (Association of Malaysian Environment-Behaviour Researchers), ABRA (Association of Behavioural Researchers on Asians) and cE-Bs (Centre for Environment-Behaviour Studies), Faculty of Architecture, Planning & Surveying, Universiti Teknologi MARA, Malaysia.  DOI: https://doi.org/10.21834/ajqol.v4i17.19

Familial Hypercholesterolaemia: An Updated Overall Management

Familial hypercholesterolaemia (FH), the commonest and serious but potentially treatable form of inherited dyslipidaemias, is characterised by severely elevated plasma low-density lipoprotein-cholesterol (LDL-C) level, which subsequently leads to premature coronary artery disease (pCAD). Effectiveness of FH early detection and treatment is supported by the outcome of several international cohort studies. Optimal FH management relies on prescription of statins either alone or together with other lipid-lowering therapies (LLT). Intensive lifestyle intervention is required in parallel with LLT, which should be commenced at diagnosis in adults and childhood. Treatment with high intensity statin should be started as soon as possible. Combination with ezetimibe and/or bile acid sequestrants is indicated if target LDL-C is not achieved. For FH patients in the very-high risk category, if their LDL-C targets are not achieved, despite being on maximally tolerated statin dose and ezetimibe, proprotein convertase subtilisin/kexin type1 inhibitor (PCSK9i) is recommended. In statin intolerance, ezetimibe alone, or in combination with PCSK9i may be considered. Clinical evaluation of response to treatment and safety are recommended to be done about 4-6 weeks following initiation of treatment. Homozygous FH (HoFH) patients should be treated with maximally tolerated intensive LLT and, when available, with lipoprotein apheresis. This review highlights the overall management, and optimal treatment combinations in FH in adults and children, newer LLT including PCSK9i, microsomal transfer protein inhibitor, allele-specific oligonucleotide to ApoB100 and PCSK9 mRNA. Family cascade screening and/or screening of high-risk individuals, is the most cost-effective way of identifying FH cases and initiating early and adequate LLT

Efficiency of Polyphenol Extraction from Artificial Honey Using C18 Cartridges and Amberlite„ XAD-2 Resin: A Comparative Study

A comparative study of the extraction efficiency of nine known polyphenols [phenolic acids (benzoic acid, dihydroxybenzoic acid, gallic acid, trans-cinnamic acid, and vanillic acid) and flavonoids (naringenin, naringin, quercetin, and rutin)] was conducted by deliberately adding the polyphenols to an artificial honey solution and performing solid phase extraction (SPE). Two SPE methods were compared: one using Amberlite XAD-2 resin and another one using a C18 cartridge. A gradient high performance liquid chromatography system with an RP18 column and photodiode array detector was utilized to analyze the extracted polyphenols. The mean percent of recovery from the C18 cartridges was 74.2%, while that from the Amberlite XAD-2 resin was 43.7%. The recoveries of vanillic acid, naringin, and rutin were excellent (>90%); however, gallic acid was not obtained when C18 cartridges were used. Additionally, the reusability of Amberlite XAD-2 resin was investigated, revealing that the mean recovery of polyphenols decreased from 43.7% (1st extraction) to 29.3% (3rd extraction). It was concluded that although Amberlite XAD-2 resin yielded a higher number of compounds, C18 cartridges gave a better extraction recovery. The lower recovery seen for the Amberlite XAD-2 resin also cannot be compensated by repeated extractions due to the gradual decrease of extraction recovery when reused

Prevalence and distributions of severely elevated low-density lipoprotein cholesterol (LDL-c) according to age, gender and clinic location among patients in the Malaysian primary care

BackgroundAdults with severely elevated low-density lipoprotein cholesterol (LDL-c) may have familial hypercholesterolaemia (FH) and are at high risk of atherosclerotic cardiovascular disease (ASCVD). The prevalence of elevated LDL-c in primary care clinics in Malaysia is not known. Therefore, this study aimed to determine the prevalence and distributions of severely elevated LDL-c among adult patients attending public primary care clinics in Malaysia.MethodsA cross-sectional study was conducted at 11 public primary care clinics in the central states of Malaysia, among adults ≥18 years old with LDL-c recorded in the electronic medical record. Sociodemographic and LDL-c data from 2018 to 2020 were extracted. Severely elevated LDL-c was defined as ≥4 mmol/L, which were further classified into: 4.0–4.9, 5.0–5.9, 6.0–6.9 and ≥ 7 mmol/L.ResultsOut of 139,702 patients, 44,374 (31.8 %) had severely elevated LDL-c of ≥4 mmol/L of which the majority were females (56.7 %). The mean (±SD) age of patients with severely elevated LDL-c was younger at 56.3 (±13.2) years compared to those with LDL-c of <4.0 mmol/L at 59.3 (±14.5) years. In terms of LDL-c levels, 30,751 (69.3 %), 10,412 (23.5 %), 2,499 (5.6 %) and 712 (1.6 %) were in the 4.0–4.9, 5.0–5.9, 6.0–6.9 and ≥ 7 mmol/L categories, respectively.ConclusionThe prevalence of severely elevated LDL-c of ≥4.0 mmol/L among adult patients in public primary care clinics was high. These patients need to be further investigated for secondary and inherited causes such as FH. Therapeutic lifestyle modification and pharmacological management are pivotal to prevent ASCVD in these patients

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field