Does epigenetic markers of HLA gene show association with coronary artery disease in Indian subjects?

BACKGROUND: DNA methylation, one of the most stable forms of epigenetic modification is associated with the development and progression of coronary artery disease (CAD). Our previously reported study on epigenome-wide microarray analysis showed significantly methylated CpG sites. Top 5 significant CpGs from HLA gene were selected and analysed by Pyrosequencing (PSQ) to determine their association with severity of CAD. METHODS: Blood samples of 50-age matched angiographically CAD positive male cases with 50 angiographically CAD negative male controls were subjected to lipid profile estimation and PSQ for methylation level analysis. Findings and subgroup analysis were evaluated by Mann-Whitney U; Kruskal-Wallis' rank test and two-way ANOVA by MedCalc (v19.6). RESULTS: Methylation levels in HLA-DQA1 for cg10217052 was 78.5 (37-85) and 76.5 (24-84); cg09411910 was 81 (72.0 to 93.0) and 81.5 (50.0 to 89.0) in cases and controls respectively. Levels in HLA-DQB1-cg03344051, were 28.88 + 9.41 for cases and 30.36 + 9.37 in controls. For HLA-DRB1-cg07889003, levels in cases and controls were 15.5 (5.00-39.00) and 10.5 (5.00-29.0); while in cg08269402 were 52 (16-65) and 42.5 (17-61) respectively. No association was observed between methylation levels and lipid profile. cg03344051, cg07889003 and cg08269402 were significantly differentiated in double or triple vessel disease (DVD or TVD) as compared to single vessel disease (SVD) suggesting an increase in the extent of methylation with the increase in CAD severity. CONCLUSION: The present study shows significant increase in the extent of methylation in 3 CpG sites in DVD/TVD cases as compared to SVD cases. Additionally, a novel site, cg07889003 identified in our discovery phase has shown association with the severity of CAD.

Results from an IFCC global survey on laboratory practices for the analysis of circulating tumor DNA.

BACKGROUND: The clinical validity of ctDNA analysis as a diagnostic, prognostic and predictive biomarker has been demonstrated in many studies. The rapid spread of tests for the analysis of ctDNA raises questions regarding their standardization and quality assurance. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices using ctDNA diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) conducted a survey among international laboratories performing ctDNA analysis. Questions on analytical techniques, test parameters, quality assurance and the reporting of findings were included. RESULTS: A total of 58 laboratories participated in the survey. The majority of the participating laboratories (87.7 %) performed testing for patient care. Most laboratories conducted their assays for lung cancer (71.9 %), followed by colorectal (52.6 %) and breast (40.4 %) cancer, and 55.4 % of the labs used ctDNA analysis for follow-up/monitoring of treatment-resistant alterations. The most frequent gene analysed was EGFR (75.8 %), followed by KRAS (65.5 %) and BRAF (56.9 %). Participation in external quality assessment programs was reported by only 45.6 % of laboratories. CONCLUSIONS: The survey indicates that molecular diagnostic methods for the analysis of ctDNA are not standardized across countries and laboratories. Furthermore, it reveals a number of differences regarding sample preparation, processing and reporting test results. Our findings indicate that ctDNA testing is being conducted without sufficient attention to analytical performance between laboratories and highlights the need for standarisation of ctDNA analysis and reporting in patient care.

Microbiome and metabolome in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease of unknown etiology, involving complex interactions between the gut microbiome and host immune response. The microbial dysbiosis is well documented in IBD and significantly influences the host metabolic pathways. Thus, a metabolomic fingerprint resulting from the influence of gut dysbiosis in IBD could aid in assessing the disease activity. PubMed, Medline, Science Direct, and Web of Science were searched for studies exploring the association between microbiome and metabolome in IBD patients in the last 5 years. Additionally, references of cited original articles and reviews were further assessed for relevant work. We provide a literature overview of the recent metabolomic studies performed on patients with IBD. The findings report alterations in the metabolite levels of these patients. We also discuss the gut dysbiosis observed in IBD and its influence on host metabolic pathways such as lipids, amino acids, short-chain fatty acids, and others. IBD, being a chronic idiopathic disease, requires routine monitoring. The available non-invasive markers have their limitations. The metabolite changes account for both dysbiosis and its influence on the host's immune response and metabolism. A metabolome approach would thus facilitate the identification of surrogate metabolite markers reflecting the disease activity.

Key methodological challenges in detecting circulating miRNAs in different biofluids.

The technological advancement in diagnostic techniques has immensely improved the capability of predicting disease progression. Yet, there is a great interest in developing newer biomarkers that can enhance disease risk prediction thereby minimising the associated morbidity and mortality. Circulating miRNAs, a non-coding RNA molecule, are critical regulators in the pathophysiology of various complex multifactorial diseases. In recent years, circulating miRNAs have been enormously studied and are considered as an emerging biomarker due to their easy accessibility, stability, and detection by sequence-specific amplification methods. However, there is a distinct lack of consensus regarding the preanalytical factors such as preferred sample selection, methodological aspects, etc that may independently or together influence the detection of circulating miRNAs resulting in erroneous expression profiles. Therefore, the present review makes an attempt to highlight the various pre-analytical and analytical factors that can potentially influence the circulating miRNA levels. Literature on circulating miRNA's stability, processing and quantitation in different biofluids along with the effect of various controllable and uncontrollable factors influencing circulating miRNA expression have been summarised in the current review.

Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort.

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340 nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6-3.9 and 7-20 nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9 months when stored at -80 °C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Molecular diagnostics of SARS-CoV-2: Findings of an international survey.

BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

Factors influencing circulating microRNAs as biomarkers for liver diseases.

MicroRNAs (microRNAs) have been implicated to play crucial roles in various liver diseases. Hepatic microRNAs are released in to the circulation in a systematic fashion, and are, therefore, being extensively explored for their role as prognostic or diagnostic markers or therapeutic targets for numerous hepatic diseases. Advantages such as disease- or tissue-specific expression, and ease of detection have implicated circulating microRNAs (c-microRNAs) as the most desirable candidate for biomarker studies. Although several studies have explored c-microRNAs in serum or plasma, consistency of detection of microRNAs remains demanding because of many biological and methodological challenges. Lack of methodological consensus has limited the universal use of c-microRNAs as potential prognostic or diagnostic disease-specific biomarkers. In this review, we have discussed pre-analytical and analytical factors that might affect c-miRNA expression and selection of appropriate data normalization strategy by incorporating endogenous reference microRNAs. This review will aid designing of better approaches and protocols for future diagnostic research on hepatic c-microRNAs.

Analytical Method Validation for Estimation of Neurotransmitters (Biogenic Monoamines) from Cerebrospinal Fluid Using High Performance Liquid Chromatography.

Biogenic amine neurotransmitters such as serotonin and dopamine are essential for signaling in both central and peripheral nervous system. Their metabolism is a multistep pathway and any defect in this results in alteration in metabolites of serotonin 5-Hydroxyindole acetic acid (5HIAA) and dopamine homovanillic acid (HVA) and 3-O-Methyl Dopa (3-OMD). Estimation of these metabolites in cerebrospinal fluid (CSF) assists in diagnosis of neurotransmitter defects. Their estimation is technically demanding and is currently available only in referral centers. We aimed to optimize a method for analysis of 5HIAA, HVA and 3-OMD. A high performance liquid chromatography (HPLC) method with electro chemical detector (ECD) was standardized for estimation. Analysis for method validation, reference range verification and clinical correlation was performed. Linearity obtained for 5-HIAA, HVA and 3-OMD was 65.35-2615.0 nmoles/l, 68.62-2745.0 nmoles/l and 236.5-4730.0 nmoles/l respectively. The coefficient of variation for internal quality controls ranged from 5 to 14% and the external proficiency testing samples (n = 16) were within peer group range. CSF metabolite levels of samples for reference range analysis overlapped with age matched ranges reported in literature. Among the 40 suspected patients analyzed for clinical testing four were found to have a neurotransmitter defect. These patients were then confirmed with molecular testing and clinical correlation. The method is validated and can be adapted in a clinical laboratory with analytical competence in HPLC.

Increased Moxifloxacin Dosing Among Patients With Multidrug-Resistant Tuberculosis With Low-Level Resistance to Moxifloxacin Did Not Improve Treatment Outcomes in a Tertiary Care Center in Mumbai, India.

BACKGROUND: Mycobacterium tuberculosis (Mtb) strains resistant to isoniazid and rifampin (multidrug-resistant tuberculosis [MDR-TB]) are increasingly reported worldwide, requiring renewed focus on the nuances of drug resistance. Patients with low-level moxifloxacin resistance may benefit from higher doses, but limited clinical data on this strategy are available. METHODS: We conducted a 5-year observational cohort study of MDR-TB patients at a tertiary care center in India. Participants with Mtb isolates resistant to isoniazid, rifampin, and moxifloxacin (at the 0.5 µg/mL threshold) were analyzed according to receipt of high-dose moxifloxacin (600 mg daily) as part of a susceptibility-guided treatment regimen. Univariable and multivariable Cox proportional hazard models assessed the relationship between high-dose moxifloxacin and unfavorable treatment outcomes. RESULTS: Of 354 participants with MDR-TB resistant to moxifloxacin, 291 (82.2%) received high-dose moxifloxacin. The majority experienced good treatment outcomes (200 [56.5%]), which was similar between groups (56.7% vs 54.0%, P = .74). Unfavorable outcomes were associated with greater extent of radiographic disease, lower initial body mass index, and concurrent treatment with fewer drugs with confirmed phenotypic susceptibility. Treatment with high-dose moxifloxacin was not associated with improved outcomes in either unadjusted (hazard ratio [HR], 1.2 [95% confidence interval {CI}, .6-2.4]) or adjusted (HR, 0.8 [95% CI, .5-1.4]) models but was associated with joint pain (HR, 3.2 [95% CI, 1.2-8.8]). CONCLUSIONS: In a large observational cohort, adding high-dose (600 mg) moxifloxacin to a drug susceptibility test-based treatment regimen for MDR-TB was associated with increased treatment-associated side effects without improving overall outcomes and should be avoided for empiric treatment of moxifloxacin-resistant MDR-TB.

Targeted exome sequencing in South Indian patients with Familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder with elevated LDL-C levels which can ultimately lead to premature Coronary Artery Disease (CAD). OBJECTIVES: In presence of limited genetic data on FH in India, the present study was aimed to determine the mutation spectrum in Indian FH patients using a targeted exome sequencing. METHODS: 54 FH cases (31 index cases + 23 extended family members) were categorized according to Dutch Lipid Clinic Network Criteria (DLCNC). Targeted exome sequencing was performed using 23 gene panel associated with lipid metabolism. RESULTS: All subjects showed the presence of family history of CAD, 38(70%) patients had corneal arcus whereas only 06(11%) subjects had xanthomas. As per the DLCNC, definite, probable, possible and unlikely FH were 48%, 30%, 11% and 11% respectively. Mutations were observed in 12 of the 23 gene panel with CETP, APOA5, EPHX2 and SREBP2 genes were identified for the first time in Indian FH patients. All 19 mutations including a novel frame-shift mutation in LDLR gene were reported for the first time in Indian FH patients. These mutations were identified in 28(52%) subjects and interestingly ∼73% of the clinically identified FH patients didn't harbour mutations in FH classical genes (LDLR, ApoB, PCSK9). CONCLUSION: This is the first study in the South Indian FH patients to perform targeted exome sequencing. Absence of mutations in the FH classical genes strongly indicates the polygenic nature of FH, further underscoring the importance of targeted exome sequencing for identifying mutations in genetically diverse Indian population.

Shortcomings on genetic testing of Familial hypercholesterolemia (FH) in India: Can we collaborate to establish Indian FH registry?

Familial hypercholesterolemia (FH) is a common autosomal dominant disorder that affects ∼1 in 250-500 individuals globally. The only prevalence study in India shows FH in 15% of patients with premature CAD in North Indians. There are only 6 genetic studies in India of the total mutations, 32% are LDLR mutations, 4% are ApoB, 2% are PCSK9 mutations and the mutational spectrum for 37% is unknown. This calls for widespread genetic screening which could help identify definite FH patients. European Atherosclerosis Society-Familial Hypercholesterolemia Studies Collaboration (EAS- FHSC) has taken an initiative to develop a worldwide registry of FH. India is also a part of the collaboration and 3 groups from Mumbai, Delhi and Chennai are actively contributing to this registry. We believe this review might help to understand the Indian scenario of FH and investigators across India can contribute in managing FH in India and further help in the detection, diagnosis and treatment.

Pharmacokinetic analysis of linezolid for multidrug resistant tuberculosis at a tertiary care centre in Mumbai, India.

Linezolid is an oxazolidinone used to treat multidrug-resistant tuberculosis (MDR-TB), including in the recently-endorsed shorter 6-month treatment regimens. Due to its narrow therapeutic index, linezolid is often either dose-adjusted or discontinued due to intolerance or toxicity during treatment, and the optimal balance between linezolid efficacy and toxicity remains unclear. India carries a significant burden of MDR-TB cases in the world, but limited information on the pharmacokinetics of linezolid and minimum inhibitory concentration (MIC) distribution is available from Indian MDR-TB patients. We enrolled participants from a tertiary care centre in Mumbai, India, treated for MDR-TB and receiving linezolid daily doses of 600 or 300 mg. Pharmacokinetic visits were scheduled between 1 and 15 months after treatment initiation to undergo intensive or sparse blood sampling. Linezolid concentration versus time data were analysed using non-linear mixed-effects modelling, with simulations to evaluate doses for different scenarios. We enrolled 183 participants (121 females), with a median age of 26 years (interquartile range [IQR] 21-35), weight 55.0 kg (IQR 45.6-65.8), and fat-free mass 38.7 kg (IQR 32.7-46.0). Linezolid pharmacokinetics was best described by a one-compartment model with first-order elimination allometrically scaled by fat-free mass and transit compartment absorption. The typical clearance value was 3.81 L/h. Simulations predicted that treatment with 300 mg daily achieves a high probability of target attainment (PTA) when linezolid MIC was ≤0.25 mg/L (61.5% of participant samples tested), while 600 mg daily would be required if MIC were 0.5 mg/L (29% of samples). While linezolid 300 mg daily is predicted to achieve effective targets for the majority of adults with MDR-TB, it failed to achieve the therapeutic target for 21% participants. A dose of 600 mg had a PTA >90% for all susceptible samples, but with a higher likelihood of exceeding toxicity thresholds (31% vs 9.6%). These data suggest potential benefit to individualized dosing taking host and microbial characteristics into account to improve the likelihood of treatment efficacy while minimizing risk of toxicity from linezolid for the treatment of MDR-TB. Further prospective evaluation in different clinical settings is urgently needed to inform safety and efficacy of these lower doses.

Cerebrospinal Fluid Biogenic Monoamine Analysis for Diagnosis of Primary Neurotransmitter Disorders.

Biogenic amine neurotransmitters metabolism is a multistep pathway with pterin and pyridoxal phosphate (vitamin B6) as cofactors. A defect in biogenic amine and cofactor metabolism and vesicular transporters result in a primary neurotransmitter disorders. These are a well-recognized groups of inherited disorders and often present with features overlapping with other neurological conditions. Their diagnosis is made by analysis of biogenic amine metabolites in cerebrospinal fluid (CSF) and other body fluids and respective enzyme assays. Many of these disorders are treatable and deficits can be reverted by timely intervention. CSF biogenic amine or cofactor metabolite analysis is one of the primary indicators of a neurotransmitter disorder. In this paper, 3 cases are reported-2 of cofactor deficiency and 1 with enzyme deficiency wherein biogenic amine estimation has assisted in diagnosis.

Current and future challenges in quality assurance in molecular diagnostics.

The development and performance of molecular genetic assays has required increasingly complex quality assurance in recent years and continues to pose new challenges. Quality management officers, as well as academic and technical personnel are confronted with new molecular genetic parameters, methods, changing regulatory environments, questions regarding appropriate validation, and quality control for these innovative assays that are increasingly applying quantification and/or multiplex formats. Yet, quality assurance and quality control guidelines are still not widely available or in some circumstances have become outdated. For these reasons, the need for solutions to provide test confidence continues to grow. In order to integrate new test procedures into existing quality assurance measures, the ISO 15189 guideline can serve as an orientation. The ISO 15189 guideline describes requirements for medical laboratories and thus includes those performing molecular diagnostics. This article gives an overview of the possibilities and challenges in quality assurance of molecular parameters and shows possible solutions.

Screening of PCSK9 and LDLR genetic variants in Familial Hypercholesterolemia (FH) patients in India.

Familial Hypercholesterolemia (FH) is an autosomal, dominant, inherited disorder characterized by severely elevated LDL-cholesterol (LDL-C) levels with high risk for Coronary Artery Disease (CAD). There are limited genetic studies especially on genes other than Low Density Lipoprotein receptor (LDLR) conducted in Indian population. Thus, our aim was to screen the entire Proprotein Convertase Subtilisin/Kexin type 9 gene (PCSK9) gene & hotspot exons 3, 4 and 9 of LDLR gene in FH cases and controls. 50 FH cases were categorized into definite, probable and possible cases according to Dutch Lipid Network Criteria (DLNC) who were gender matched with 50 healthy controls. All 12 exons of PCSK9, and hotspot exons 3, 4 & 9 of LDLR gene were screened through High Resolution Melt (HRM) curve analysis. Enzyme linked immunosorbent assay was performed to measure circulating PCSK9 levels. Total cholesterol and LDL-C were significantly high in all three groups of cases. Total 8 nonpathogenic variants in exon 1, 5, 7 and 9 of the PCSK9 gene were detected. In LDLR gene, 3 known pathogenic and 1 benign variant were found in exon 3 & 4. In FH cases, PCSK9 levels were significantly high compared to controls (P = 0.0001), and were directly correlated to LDL-C (P = 0.0001) and Total Cholesterol (P = 0.0001). Our study is first to screen the entire PCSK9 gene in western part of India. Since no pathogenic variants were identified, it is possible that PCSK9 variants are clinically less relevant. However, 3 known pathogenic variants were found in the LDLR gene. These findings support our understanding of the genetic spectrum of FH in India.

Optimization of infliximab therapy in inflammatory bowel disease using a dashboard approach-an Indian experience.

PURPOSE: Infliximab (IFX) therapy in inflammatory bowel disease (IBD) is associated with loss of response in half the patients, due to complex pharmacokinetic and immunological factors. Dashboard's Bayesian algorithms use information from model and individual multivariate determinants of IFX concentration and can predict dose and dosing interval. AIM: To compare measured IFX concentrations in our laboratory with values predicted by iDose dashboard system and report its efficacy in managing patients not responding to conventional dosing schedule. METHOD: Clinical history, demographic details, and laboratory findings such as albumin and C-reactive protein (CRP) data of IBD patients (n = 30; median age 23 years (IQR: 14.25 - 33.5)) referred for IFX drug monitoring in our laboratory from November 2017 to November 2019 were entered in iDose software. The IFX concentration predicted by iDose based on this information was compared with that measured in our laboratory. In addition, a prospective dashboard-guided dosing was prescribed in 11 of these 30 patients not responding to conventional dosing and was followed to assess their clinical outcome. RESULT: IFX monitoring in our 30 patients had shown therapeutic concentration in 12, supratherapeutic in 2 and subtherapeutic concentration in 16 patients. The iDose predicted concentration showed concordance in 21 of these 30 patients. Of 11 patients managed with iDose-assisted prospective dosing, 8 achieved clinical remission, 2 showed partial response, and one developed antibodies. CONCLUSION: Retrospective data analysis showed concordance between laboratory measured and iDose-predicted IFX level in 70% of patients. iDose-assisted management achieved clinical remission and cost reduction.

Association of Genetic Variants with Hyperhomocysteinemia in Indian Patients with Thrombosis.

Hyperhomocysteinemia known to be associated with increased thrombotic tendency has been considered as a risk factor for coronary artery disease, atherosclerosis, venous thrombosis, and stroke. There are three main genes MTHFR, cystathionine beta-synthase (CBS) and methionine synthase (MS) and it's genetic variant that are known to influence the homocysteine metabolism leading to hyperhomocysteinemia. There is scarcity of Indian data on hyperhomocysteinemia and genetics variants in patients with thrombosis. Hence the objective of present study was to determine MTHFR, CBS, and MS genetic variants in thrombosis patients from Indian population. Genetic variant analysis was performed on thrombosis patients to detect MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), MS A2756G (rs1805087) and CBS T833C (rs5742905) mutations. The mutant allele frequencies of MTHFR 677T, MTHFR 1298C, MS2756G and CBS 833C were observed to be 16.1%, 37.5%, 34.1% and 5.8% respectively. MTHFR 677TT genotype was observed to be significantly associated with elevated homocysteine (Hcy) levels (64.65 µmol/L) alleles as compared to CC alleles (32.43 µmol/L) and CT alleles (30.54 µmol/L). MTHFR A1298C, MS A2756G and CBS T833C genotypes did not showed significant association with higher Hcy levels. Thus, in Indian patients with thrombosis only MTHFR T677T genotype was observed to be significantly associated with hyperhomocysteinemia.