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.

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.

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.

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.

Circulating miRNA-33: a potential biomarker in patients with coronary artery disease.

BACKGROUND: Circulating microRNAs (miRNA) are present in body fluids in stable, cell-free form. Likewise, these miRNAs can be identified in various stages of coronary artery disease (CAD) such as inflammation, endothelial dysfunction, proliferation and atherosclerosis among others. miRNA expression levels can be identified. AIMS AND OBJECTIVES: To determine the expression of circulating miRNAs (miR-126, miR-92, miR-33, miR-145 and miR-155) in CAD patients of Indian origin. MATERIAL AND METHODS: miRNA profiling analysis in blood plasma was performed by quantitative real-time-PCR (qRT-PCR) in 60 angiographically verified subjects including 30 CAD patients and 30 age- and gender-matched controls. Association between the expression of all five circulating miRNAs and clinical characteristics of patients with CAD were analysed using Medcalc statistics. The severity of CAD was assessed using SYNTAX score (SS). RESULTS: Expression of plasma miR-33 increased by 2.9 folds in CAD patients than in control group (p value ≥0.002) also it was found that miR-33 expression levels in mild cases (SS: ≤22) were significantly higher than CAD controls. There was a modest negative correlation between miR-33 and total cholesterol/high density lipoprotein ratio, triglycerides and very low density lipoprotein. CONCLUSION: The study reports a significant association between increased levels of plasma miR-33 and CAD. Thus, plasma miR-33 appears to be a promising non-invasive biomarker, but requires further validation in a large cohort.

Reference intervals for 33 biochemical analytes in healthy Indian population: C-RIDL IFCC initiative.

Background In 2011, the IFCC Committee on Reference Intervals and Decision Limits (C-RIDL) initiated a worldwide multicenter study on references values facilitating the implementation of country-specific reference intervals (RIs). There has been no well-designed RI study in India. This study aims to derive RIs for 33 major biochemical analytes in carefully selected healthy Indians as defined in C-RIDL protocol. Methods A total of 512 healthy Indians were recruited. Sera collected from overnight fasting blood samples were measured collectively for the analytes. Multiple regression analysis (MRA) and nested analysis of variance (ANOVA) were used to identify the potential sources of variation (SV) of test results. RI were derived by both parametric and non-parametric methods for comparison. The need for secondary exclusion by latent abnormal values exclusion (LAVE) method was examined. Results MRA results indicated that both age and BMI were apparent SV for many analytes in both sexes. ANOVA revealed that partition of RIs by gender and age was required for 17 analytes (TC, HDL-C, TG, hsCRP, ALB, AST, ALT, ALP, GGT, TBil, Urea, CRE, UA, Fe, TTR, CK and IgM) and 5 (Glu, ALB, TC, ALP and Urea), respectively. RIs by parametric method were generally narrower than by non-parametric method, reflecting distorted peripheral distributions of test results. The LAVE method had no appreciable effect on RIs possibly due to inconsistency among abnormal values of related analytes. Conclusions This study has for the first time provided comprehensive RIs information in healthy Indians. The final RIs adopted were those derived by parametric method without LAVE procedure.

Preemptive NUDT15 genotyping: redefining the management of patients with thiopurine-induced toxicity.

BACKGROUND: Thiopurine methyltransferase (TPMT) gene variants have achieved limited success in predicting the outcome of thiopurine therapy, which shows wide inter-individual variations. The literature indicates a strong association between the NUDT15 gene variant and thiopurine-induced toxicity in Asian patients. The present study intends to explore the role of the NUDT15 variant (C415T) in Indian patients on thiopurine therapy. METHODS: NUDT15 and TPMT genotyping were performed using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) and the restriction fragment length polymorphism (RFLP) technique. RESULTS: Of 370 samples received for TPMT testing, 206 samples were available for NUDT15 genotyping. The NUDT15 risk allele frequency was 10.7%, with the frequency of wild, heterozygous and mutant genotypes being 80.6%, 17.5% and 1.9%, respectively. TPMT variants were seen in 13 of 370 (3.5%) patients, whereas the NUDT15 variant was seen in 40 of 206 (19.4%) patients. Thiopurine-induced toxicity information was available for 101 patients, among whom 10 developed leukopenia and all harbored the NUDT15 variant (p<0.0001). NUDT15 was clinically more relevant than TPMT in terms of sensitivity and specificity, as well as with a statistically significant difference in thiopurine dose requirement for patients with the NUDT15 variant. CONCLUSIONS: A preemptive NUDT15 genotyping approach can therefore help identify high-risk patients (NUDT15 C415T positive) who could benefit from thiopurine dose reduction, thereby preventing fatal thiopurine-induced toxicity.

A global multicenter study on reference values: 2. Exploration of sources of variation across the countries.

OBJECTIVES: The intent of this study, based on a global multicenter study of reference values (RVs) for serum analytes was to explore biological sources of variation (SVs) of the RVs among 12 countries around the world. METHODS: As described in the first part of this paper, RVs of 50 major serum analytes from 13,396 healthy individuals living in 12 countries were obtained. Analyzed in this study were 23 clinical chemistry analytes and 8 analytes measured by immunoturbidimetry. Multiple regression analysis was performed for each gender, country by country, analyte by analyte, by setting four major SVs (age, BMI, and levels of drinking and smoking) as a fixed set of explanatory variables. For analytes with skewed distributions, log-transformation was applied. The association of each source of variation with RVs was expressed as the partial correlation coefficient (rp). RESULTS: Obvious gender and age-related changes in the RVs were observed in many analytes, almost consistently between countries. Compilation of age-related variations of RVs after adjusting for between-country differences revealed peculiar patterns specific to each analyte. Judged fromthe rp, BMI related changes were observed for many nutritional and inflammatory markers in almost all countries. However, the slope of linear regression of BMI vs. RV differed greatly among countries for some analytes. Alcohol and smoking-related changes were observed less conspicuously in a limited number of analytes. CONCLUSION: The features of sex, age, alcohol, and smoking-related changes in RVs of the analytes were largely comparable worldwide. The finding of differences in BMI-related changes among countries in some analytes is quite relevant to understanding ethnic differences in susceptibility to nutritionally related diseases.

Nucleoside diphosphate-linked moiety X-type motif 15 C415T variant as a predictor for thiopurine-induced toxicity in Indian patients.

BACKGROUND AND AIM: Interindividual variation seen in the thiopurine metabolism is attributed to the genetic variant in thiopurine methyltransferase (TPMT) gene leading to myelosuppression. In Asians, the thiopurine-induced toxicity is not completely explained by TPMT variants. Literature indicates that a newer genetic variant in nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene is associated with thiopurine intolerance. We aimed to determine the risk allele frequency of NUDT15 genetic variant and its association with thiopurine-induced toxicity in Indian patients. METHODS: In this pilot study, 69 patients on thiopurine therapy were analyzed. The frequencies of thiopurine-induced leukopenia were recorded. NUDT15 (C415T) and TPMT (*2, *3A, *3B, and *3C) genotyping was performed using amplification refractory mutation system-polymerase chain reaction and restriction fragment length polymorphism technique. Results were validated by DNA sequencing. RESULTS: The NUDT15 CC, CT, and TT genotypes were found to be 86.9%, 11.5%, and 1.5%, respectively, whereas TPMT genetic variants were absent. Of 60 patients without NUDT15 variant, none developed leukopenia, whereas of nine patients with NUDT15 variant, six developed leukopenia (P-value < 0.0001). The mean thiopurine dose of 1.01 and 0.73 mg/kg/day for patients with wild and mutant NUDT15 alleles, respectively, was statistically significant (P < 0.01). The sensitivity and specificity for NUDT15 variant were 100% and 95.2%, respectively. CONCLUSIONS: The NUDT15 risk allele frequency was 7.2%. There are 6/69 (8.7%) patients who developed leukopenia and harbored NUDT15 variant, thus showing a strong association for thiopurine-induced toxicity. Hence, NUDT15 genotyping may be considered before thiopurine therapy in Indian patients.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases.

BACKGROUND: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD. METHODS: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing. RESULTS: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing. CONCLUSION: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

Troponin T and Heart Type Fatty Acid Binding Protein (h-Fabp) as Biomarkers in Patients Presenting with Chest Pain.

Acute coronary syndrome (ACS) is a term for a range of clinical signs and symptoms suggestive of myocardial ischemia. It results in functional and structural changes and ultimately releasing protein from injured cardiomyocytes. These cardiac markers play a major role in diagnosis and prognosis of ACS. This study aims to assess the efficacy of heart type fatty acid binding protein (h-FABP) as a marker for ACS along with the routinely used hs-TropT. In our observational study, plasma h-FABP (cut-off 6.32 ng/ml) and routinely done hs-Trop T (cutoff 0.1 and 0.014 ng/ml) were estimated by immunometric laboratory assays in 88 patients with acute chest pain. Based on the clinical and laboratory test findings the patients were grouped into ACS (n = 41) and non-ACS (n = 47). The diagnostic sensitivity, specificity, NPV, PPV and ROC curve at 95 % CI were determined. Sensitivity of hs-TropT (0.1 ng/ml), hs-TropT (0.014 ng/ml) and h-FABP were 53, 86 and 78 % respectively and specificity for the same were 98, 73 and 70 % respectively. Sensitivity, specificity and NPV calculated for a cut-off combination of hs-TropT 0.014 ng/ml and h-FABP was 100, 51 and 100 % respectively. These results were substantiated by ROC analysis. Measurement of plasma h-FABP and hs-TropT together on admission appears to be more precise predictor of ACS rather than either hs-Trop T or h-FABP.

Role of thrombomodulin gene in Indian population with coronary artery disease.

CONTEXT: Thrombomodulin (TM), a natural anticoagulant have been implicated in the pathogenesis of coronary artery disease (CAD) thus emphasizing its potential role as a biomarker. OBJECTIVES: To investigate the role of the TM genetic variants and soluble TM (sTM) plasma levels in Indian population with CAD. MATERIALS AND METHODS: This case-control study involved genotyping of the entire TM gene and sTM levels estimation in 266 subjects. RESULTS: None of the four TM genetic variants identified significantly increased CAD risk in the study population. However, further subgroup analysis revealed that in subjects ≤49 years, C1418T variant (Ala455Val substitution) was significantly associated with CAD. CONCLUSION: The increased CAD risk in subjects ≤49 years due to TM Ala455Val substitution is a promising finding. Further validation on large Indian cohorts is required in order to screen asymptomatic young subjects for CAD risk and to establish the clinical utility of Ala455Val substitution.

Cardiovascular disease in India.

Cardiovascular disease (CVD) is one of the leading cause of mortality in India. It is estimated that 23.6 million CVD cases will be reported in subjects younger than 40 years of age by 2015, suggesting that young Indians are at higher cardiac risk. Evaluation of biomarkers in acute coronary syndrome (ACS) and at various stages of the disease such as inflammation, ischemia, and heart failure would indeed help to assess cardiac risk in Indian subjects. Identification of newer genetic markers through the candidate and/or genome-wide association approach would prove to be beneficial in developing a diagnostic assay for screening young asymptomatic Indian subjects.

Prevalence of metabolic syndrome in urban India.

Background. Metabolic syndrome (MS) is characterised by a constellation of individual risk factors of cardiovascular disease. Materials and Methods. The current study was a population-based survey of cohort of subjects in the metropolitan city of Mumbai. A total of 548 subjects, who attended the CARDIAC evaluation camp, were recruited in the study. Participants with complete fasting lipid profiles, blood glucose, and known cardiac risk markers were evaluated. Results. On applying modified NCEP ATP III, we found out that nearly 95% of the subjects had at least one abnormal parameter. We found the prevalence of MS in our study population to be 19.52%. The prevalence of MS in males was almost double than females (P = .008). The overall prevalence of BMI (>23 kg/m(2)) was 79.01%. Increased hypertriglyceridemia and decreased levels of HDL-C were found to be more in males (P < .0001). Conclusion. The low percentage of subjects with normal and controlled parameters suggests that there is a need for awareness programs and lifestyle interventions for the prevention and control of MS.

Design of Allele Specific PCR for Rapid Detection of CYP3A5 (A6986G) and Mdr-1 (C3435T) Polymorphisms.

Single nucleotide polymorphisms in CYP3A5 (A6986G) and MDR-1 (C3435T) genes have been shown to be associated with the pharmacokinetics of tacrolimus in case of renal transplant recipients. Knowing these genotypes of the recipients before undergoing transplantation, is therefore essential for physicians to adjust the starting dose of tacrolimus in order to avoid drug induced nephrotoxicity. We have designed an allele specific PCR method for easier and rapid detection of these polymorphisms. 20 Indian renal transplant recipients on tacrolimus who developed nephrotoxicity within 1 month of transplantation and 58 Indian non-transplant subjects having the risk factors for kidney disease i.e. hypertension or diabetes or the family history of these, have been studied for these SNPs by allele specific PCR method. The data suggest that the heterozygosity of CYP3A5 and mutant allele frequency of MDR-1 SNP is higher in transplant patients as well as in general population.

TPMT and DPD polymorphisms: Efficient screening method for Indian patients considering taking Thiopurine and 5-FU drugs.

INTRODUCTION: Development of DNA-based tests for TPMT/DPD polymorphisms can help clinicians and patients to make important decisions about cancer treatment. Also, due to lack of Indian data, we aimed at the development and validation of these tests in Indian patients. MATERIALS AND METHODS: Molecular assays were used for identifying TPMT/DPD variations; validated by DNA sequencing. RESULTS: Molecular assays have been used for screening TPMT*2, *3A, *3B, *3C alleles and IVS14+1(G-->A) in DPD gene. A patient, exhibiting neutropenia on 6-MP was observed to be G460A-homozygote, while, two Acute Lymphoblastic Leukemia (ALL) patients with side-effects exhibited wild-type alleles. Two patients showing 6-MP side-effects and responding well to the same drug at later stage also carried wild-type alleles. DISCUSSION: G460A homozygosity in a patient allowed clinicians to stop 6-MP treatment, improving patient's health status. Two ALL patients showing side-effects were wild-type, indicating presence of unidentified rare variations. Two patients with wild-type allele showed side-effects during 6-MP treatment, but responded well to same drug at later stage, suggesting side-effects to be attributable to multiple biological and environmental processes. Absence of IVS14+1(G-->A) in DPD gene will not exclude possibility of another mutation. CONCLUSION: Molecular assays for determining common TPMT/DPD variations, can provide accurate diagnosis and efficient therapies in future clinical studies.