DRD4 and DAT1 VNTR Genotyping in Children with Attention Deficit Hyperactivity Disorder.

The dopamine receptor-D4 and the dopamine transporter have been investigated for their role in attention deficit hyperactivity disorder (ADHD) in children. Reports of their genetic association with ADHD have shown mixed results. The aim of the study was to evaluate the association of variable number tandem repeats (VNTRs) of the DRD4 and DAT1 genes with ADHD in children. A pilot 1:1 case control study, with 44 clinically confirmed ADHD cases and 44 age/gender matched healthy controls, was conducted at a tertiary care centre in Mumbai. Variable number tandem repeats of DRD4 exon 3, DAT1 intron 8 and 3'UTR were genotyped by PCR-AGE. Several allele repeats of the genes were observed in the screened subjects. Statistical significance was observed for the 10R/10R genotype of the DAT1 3'UTR VNTR between cases and controls.

Association between genetic loci linked to HDL-C levels and Indian patients with CAD: a pilot study.

OBJECTIVE: To examine the association between loci linked to high-density lipoprotein cholesterol (HDL-C) levels and coronary artery disease (CAD). METHODS: A pilot study consisting of age-matched and gender-matched angiographically confirmed CAD cases (n=150) and non-CAD controls (n=150) was performed to test an association. Illumina's Human Cardio-Metabo BeadChip containing 3112 variants associated with HDL-C levels was used for genotyping. RESULTS: A preliminary analysis identified 36 variants from 16 genes that were statistically significant (p<0.05) between cases and controls. However, none of the variants remained statistically significant after correction for multiple testing. Besides, variants rs11039159 (MADD), rs749067 (MADD), rs367070 (LILRA3) and rs330921 (PPP1R3B) showed modest association with HDL-C levels. CONCLUSIONS: None of the HDL-C associated loci included in this study were found to be a significant risk factor for CAD. However, the study could replicate the findings of four variants influencing HDL-C levels.

Genetic determinants of lipid-lowering response to atorvastatin therapy in an Indian population.

WHAT IS KNOWN AND OBJECTIVE: Statins form the backbone of lipid-lowering therapy for the prevention of cardiovascular disease. However, there is large interindividual variability in clinical response to statin treatment. Several gene variants that can be aligned to either the pharmacokinetics or pharmacodynamics of statin have been proposed as potentially important determinants of statin response. We aimed to study the association of known variations in SLCO1B1, CYP3A4, ABCB1, CYP3A5, ABCG5 and CYP7A1 genes with lipid levels in response to atorvastatin therapy. METHODS: Genotypes were determined using multiplex allele-specific polymerase chain reaction in 177 Indian patients, treated with 10 mg of atorvastatin for 8 weeks. Low-density lipoprotein-cholesterol (LDL-C) levels were recorded at baseline and after 8 weeks of atorvastatin treatment. RESULTS AND DISCUSSION: A total of 177 hypercholesterolaemic patients were genotyped to study genetic determinants of atorvastatin response. The genotype distribution for all polymorphisms investigated was in Hardy-Weinberg equilibrium. In our study, patients with wild-type genotypes of CYP7A1 (rs3808607), CYP3A4 (rs2740574), SLCO1B1 (rs2306283) and variant allele-carrying genotype of ABCB1 (rs2032582, rs1045642) showed significantly greater LDL-cholesterol reductions in response to atorvastatin therapy. WHAT IS NEW AND CONCLUSION: The variable response to atorvastatin therapy in terms of LDL-cholesterol lowering due to genetic variations in CYP7A1, CYP3A4, SLCO1B1 and ABCB1 is a promising finding. Further validation in large Indian cohorts is required before it can be assessed for clinical utility.

Urinary Glycosaminoglycan Estimation as a Routine Clinical Service.

Mucopolysaccharidoses, a group of inherited disorders are associated with defects in glycosaminoglycan metabolism. Thus, assessment of urinary glycosaminoglycan is used as a screening test for mucopolysaccharidoses. The detection methods range from qualitative spot tests to quantification using metachromatic dyes. In our laboratory we optimized a spectrophotometric quantitative method using a metachromatic dye, dimethylmethylene blue. Heparan sulfate was used for quantification. The glycosaminoglycan-dye complex showed a marked shift in color with increase in concentration. The color complex was quantified at 520 nm. The method was linear from 10-89 mg/L. An age matched normal range was obtained in 177 healthy individuals, grouped in 8 different age groups from neonates to adults. Urinary glycosaminoglycan concentration varied distinctly amongst the study population wherein the lowest range in healthy neonates was more than 3 times the upper limit of healthy adults. Urine samples from 10 patients with mucopolysaccharidoses were also included in the study for clinical validation. The method qualified both analytical and clinical validation and was found to be simple, robust and ideal to be offered as a screening test for mucopplysaccharidoses in a routine clinical chemistry laboratory.

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.

Unusual dendritic keratitis.

Bilateral pseudo-dendritic keratitis in infancy can be due to tyrosinemia, a rare metabolic disorder. Ocular involvement may be the earliest presenting manifestation of this disease. Early diagnosis is essential because dietary modifications can result in complete reversal of the manifestations of this disorder. This disease must be suspected in all cases of non-responsive dendritic keratitis in the pediatric age group, especially if it is associated with cutaneous lesions such as patmoplantar keratosis. Serum tyrosine levels must be done in these cases.

Apolipoprotein E polymorphism and coronary heart disease.

Apolipoprotein E is a constituent of various lipoproteins and plays an important role in the transport of cholesterol and other lipids among cells of various tissues. The gene is polymorphic with three alleles (epsilon2, epsilon3, and epsilon4) coding for isoforms E2, E3, and E4 and having different binding affinities for the apo E receptors. While the epsilon2 allele is associated with elevated triglyceride levels, epsilon4 allele is associated with increased cholesterol levels. Though several studies support the role of apo E polymorphism in CHD either directly or indirectly via its influence on lipid and lipoprotein levels, there are some studies, which show no association. With the increasing incidence of CHD among Indians, it becomes imperative to identify genetic markers that may predispose individuals to coronary events. It would be of importance to determine if apo E gene will become a usefuladjunct to assess cardiovascular risk profile when performing genetic studies in families.

Coronary heart disease, hypertension, and angiotensinogen gene variants in Indian population.

Gene encoding components of the renin angiotensin system (RAS) have been implicated with the increased risk of cardiovascular disease (CVD). Two variants of the angiotensinogen (AGT) gene, M235T and T174M, have been shown to be associated with increased risk of hypertension. In the present study, we examined the association of these two polymorphisms and their synergistic interaction with the angiotensin I-converting enzyme (ACE) deletion homozygote genotype (D/D) on subjects with coronary heart disease (CHD) and hypertension. We studied 131 healthy individuals, 141 angiographically verified CHD patients, and 159 hypertensive subjects. The identification of the ACE and AGT gene polymorphisms was carried out using a PCR-based restriction endonuclease digestion method. There was no significant difference in the distribution of the M235T and T174M variants between the two test groups and the control group. Association was also not seen when analysis was carried out in patients when subgrouped according to the extent of the severity of the disease. In addition, the risk was not restricted to subjects carrying the D allele of the ACE gene and T235T of AGT. M235T and T174M variants do not contribute to the increased risk of CHD or hypertension in the Indian population.

Molecular diagnosis of cystic fibrosis in Indian patients--a preliminary report.

AIM: The aim of the study was to screen for the common deltaF508 mutation and the poly T polymorphism and to determine their frequency in the cystic fibrosis transmembrane conductance regulator (CFTR) gene among the suspected CF cases referred to our clinical care centre for sweat chloride tests. METHODOLOGY: Sweat and EDTA blood samples were obtained from 23 clinically suspected cystic fibrosis (CF) cases. Sweat was estimated by pilocarpine iontophoresis procedure. Poly T polymorphism was detected by the multiplex-PCR based on ARMSTM technique and deltaF508 mutation by PCR-mediated site-directed mutagenesis method. RESULTS: Five cases, mainly with respiratory abnormalities and followed by steatorrhea had elevated sweat chloride levels (> 60 mmol/l), three of them, each with nutritional, respiratory and pancreatic abnormalities were borderline (40-60 mmol/l) and the remaining 15 clinically suspected CF cases had normal sweat chloride levels (< 40 mmol/l). The 9T variant was frequently observed (75%) in cases with elevated sweat chloride, including those exhibiting borderline values; with no 5T variant. The 7T was the most common variant (77%) observed in the cases with normal sweat chloride, with only one 5T variant (33%). Of the five cases with high sweat chloride, four cases were homozygous for deltaF508, whereas one was heterozygous with borderline sweat chloride, thus showing an overall frequency of 56.25% in the CF chromosome. DeltaF508 was found to be present with the 9T variant in all the instances. CONCLUSION: The presence of the 9T variant along with elevated sweat chloride levels can be used to predict a high risk of the individual harboring the severe deltaF508 mutation. It would be advisable to test for to the deltaF508 mutation along with the sweat chloride estimation in all the critically suspected CF cases diagnose CF with a higher degree certainty.

Methylenetetrahydrofolate reductase gene mutation and hyperhomocysteinemia as a risk factor for coronary heart disease in the Indian population.

OBJECTIVE: The screening and therapeutic guidelines for the management of lipid abnormalities are reasonably well established. However, other risk factors like hyperhomocysteinemia (HCA) and single nucleotide polymorphisms involving the angiotensin converting enzyme (ACE) and angiotensinogen genes, various clotting factors etc., have yet to be established firmly as other causative factors of atherothrombotic disease. Our study was aimed at finding the relationship between HCA, folate, vitamins B12 levels, and mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS) genes. METHODS: We studied 230 subjects, which included patients with angiographically documented coronary heart disease (CHD) (n=115) and controls (n=115) with no history of CHD. RESULTS: Elevated levels of plasma homocysteine, above 18 nmoles/ml, were detected in 19.13% and 18.26% of our patients and controls, respectively. Homocysteine was significantly correlated to Apo A1 (r=0.51, p < 0.05) and Apo B (r=0.49, p < 0.05). The heterozygous MTHFR mutation was found to be 54.5% (12/22) in our patients with HCA. Of these, 31.8% (7/22) were deficient for plasma folate. Heterozygosity for T833C mutation in the CBS gene was observed in 9.99% (2/22) of our patients with HCA. Both these patients were also deficient for plasma folate and vitamin B12. CONCLUSION: In our study, heterozygosity for the thermolabile MTHFR mutation was found to be associated with hyperhomocysteinemia (HCA). This genetic predisposition to HCA could be risk factor for CHD and can be correlated with vitamin supplementation. To the best of our knowledge this is the first report from India on plasma homocysteine levels and its genetic aspect in patients with CHD.

ACE and AT1R gene polymorphisms and hypertension in Indian population.

The renin angiotensin system (RAS) controls intrarenal blood pressure and sodium balance, and is an important target for antihypertensive therapy. Several polymorphisms have been identified within genes encoding RAS that may contribute to the development of elevated blood pressure. The relevance of these polymorphisms in hypertension remains controversial. In this study we have examined 105 hypertensive subjects and 192 controls from the Indian population for I/D polymorphism of angiotensin I converting enzyme (ACE) and A(1166)C polymorphism of angiotensin II type I receptor (AT1R) genes by polymerase chain reaction (PCR) and PCR-based restriction enzyme analysis method, respectively. There was no significant difference in the distribution of ACE (I/I, I/D, and D/D) and AT1R (A/A and A/C) genotypes between controls and hypertensive subjects. D allele was significantly associated with an early onset of hypertension and although nonsignificant, the frequency was high in subjects with family history of cardiovascular disorders. C(1166) allele of AT1R did not correlate with the age of onset of hypertension and the frequency was low in subjects with family history. Thus no association was found between ACE and AT1R genotypes and hypertension. However the D allele can be used as a predictor of risk of hypertension in the Indian population.

Identification of two LDL receptor mutations causing familial hypercholesterolemia in Indian subjects.

Familial hypercholesterolemia (FH) is a genetic disorder caused by numerous mutations in the low-density lipoprotein receptor (LDLR) gene. Mutational analyses of Indians in South Africa suggest the possibility of a high frequency of FH in India. This study aimed at identifying mutations in exons 3, 4, 9, and 14 of the LDLR gene among Indians and at eventually developing population-directed molecular-based screening assays. DNA samples from 25 hypercholesterolemic patients with clinical features of FH and 25 normal controls were analyzed for four known point mutations: W66G (exon 3), E207K (exon 4), E387K (exon 9), and P664L (exon 14), which are those most reported among Indian immigrants in South Africa. Subsequently, samples were screened for other mutations by modified heteroduplex analysis in these exons. Point mutations predicted to be common among Indians were absent in the samples analyzed. Heteroduplex analysis and sequencing revealed the presence of novel insertion mutations in two patients. Both mutations are single-nucleotide "G" insertions at position 242 in exon 3 and position 397 in exon 4. The observed mutations are predicted to cause a translational frameshift, encoding truncated LDLR proteins due to premature termination. The mutant proteins are likely to be degraded intracellularly and, therefore, are pathogenic.

Genes of renin angiotensin system and coronary heart disease.

Coronary constriction, proliferation of smooth muscle cells and arrhythmia are involved in the pathophysiology of coronary heart disease and its complications such as myocardial infarction and sudden death. All these effects are favoured by high angiotensin II levels. Angiotensin II is the main effector molecule of the renin angiotensin system and it acts through angiotensin II type receptors. Genetically determined differences in the expression of the components of this system could adversely affect angiotensin II concentration and subsequently heart. Consequently each component of this system represents a potential candidate in the etiology of cardiovascular disease. In this article we review the variation of the angiotensin I converting enzyme, angiotensin II type I receptor and angiotensinogen genes and their association with cardiovascular disease.

Molecular basis of familial hypercholesterolemia: An Indian experience.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by a multitude of low-density lipoprotein (LDL) receptor gene mutations. The LDL receptor is a cell surface trans-membrane protein that mediates the uptake & lysosomal degradation of plasma LDI., thereby providing cholesterol to cells. Affected individuals have elevated plasma levels of LDL, which causes premature coronary atherosclerosis. FH has an estimated worldwide prevalence of 0.2%. In some subpopulations there is an increased frequency of FH and specific LDL receptor mutations are found to be common due to 'founder gene effect'. Overall, more than 300 naturally occurring LDL receptor mutations have been described. To data upto ten LDL receptor gene mutations have been identified in Indians in South Africa, suggesting increased incidence of FH among Indians. Most mutations have occurred at CpG dinucleotide, a mutational hotspot in human genetic disease. In our study in 25 hypercholesterolemic subjects we have identified two novel insertion mutations in two patients. But the mutations underlying FH are still undefined in the majority of cases. Mutational heterogeneity on the other-hand has complicated disease diagnosis at DNA level. These findings warrant application of a generalized mutation screening method in search for new LDL receptor gene defects.

Angiotensin converting enzyme gene polymorphism in coronary artery disease: the Indian scenario.

Angiotensin converting enzyme is a key component of the renin angiotensin system that plays an important role in cardiovascular regulation. It seems to modulate cardiovascular growth by virtue of its role in the conversion of angiotensin I to angiotensin II and degradation of kinins. A deletion polymorphism localized in intron 16 of the human angiotensin converting enzyme gene, corresponding to a 287 bp long Alu repetitive sequence, was found to be associated with increased risk of myocardial infarction in various subgroups, including European, French and Japanese coronary patients. This angiotensin converting enzyme gene I/D polymorphism was examined by the polymerase chain reaction in a cross-sectional study of 201 healthy Indian subjects and 150 patients (angiographically proven cases of coronary artery disease) whose serum angiotensin converting enzyme levels were concomitantly measured. The D/D, I/D and I/I genotypes were found in 20.66%, 46.66% and 32.66% of the Indian coronary heart disease patients and in 23.38%, 49.75% and 26.86% of controls respectively. One of the reasons for not finding an association between the D allele and coronary artery disease in this study could be the ethnic heterogeneity and disease status heterogeneity among the patients and controls. However the phenotypic variance of serum angiotensin converting enzyme levels is strongly influenced by this polymorphism. In the Indian population, the angiotensin converting enzyme gene I/D polymorphism is not associated with risk for coronary artery disease although it is associated with plasma angiotensin converting enzyme activity. Hence the angiotensin converting enzyme gene I/D polymorphism does not seem to be a useful marker for coronary artery disease in the Indian population.

Characterisation of the canine cardiac sarcolemma in experimental myocardial ischemia.

The cardiac sarcolemma was characterized in 13 normal and 11 ischemic dog hearts by enzyme analysis and compositional assays. Significant decreases in the activities of the sodium-potassium and calcium pumps and structural compositional disturbances were observed in ischemia. High concentrations of oleic acid, a fatty acid and palmitoyl carnitine, a fatty acid intermediate caused inhibition of the enzyme pump activities of the normal sarcolemma. Thus, ischemia results in the functional impairment of the sarcolemma. Accumulation of fatty acid and fatty acid intermediates, occurring in myocardial ischemia, could be an underlying mechanism.

Renal involvement in multiple myeloma.

The present study is a retrospective chart analysis of 33 patients who satisfied the diagnostic criteria of multiple myeloma. Sixteen (49.5%) of these 33 patients developed renal failure at some point in time. The mean age +/- 1SD of patients who developed renal failure was 59.2 +/- 13 years (range 34-85 years). There were 12 males and 4 females. The precipitating factors for renal failure were dehydration (12.5%), hypercalcemia (62.5%) and use of non-steroidal antiinflammatory drugs (6.2%). Hypercalcemia was observed in 10 of the 16 patients who developed renal failure while it was seen in only 4 of the 17 cases who did not develop renal failure (relative risk 5.4). In 11 (68.7%) patients, the renal function improved with hydration, treatment of hypercalcemia and chemotherapy. The 1 and 3 year actuarial survival of patients with renal failure and multiple myeloma was 87% and 74% respectively.