Kunal Ray

Wilson's disease: an update

Wilsons disease (WD) is an inborn error of copper metabolism caused by a mutation to the copper-transporting gene ATP7B. The disease has an autosomal recessive mode of inheritance, and is characterized by excessive copper deposition, predominantly in the liver and brain. Diagnosis of the condition depends primarily on clinical features, biochemical parameters and the presence of the Kayser–Fleischer ring, and a new diagnostic scoring system has recently been proposed. Mutations in ATP7B can occur anywhere along the entire 21 exons, which makes the identification of gene defects particularly challenging. Identification of carriers and presymptomatic family members of affected individuals is achieved by polymerase-chain-reaction-based marker analysis. The traditional treatment for WD is based on copper chelation with agents such as D-penicillamine, but use of this drug has been questioned because of reported side effects. The use of agents such as trientine and ammonium tetrathiomolybdate has been advocated, although results of long-term trials are awaited. In selected cases, orthotropic hepatic transplantation can reverse the basic metabolic abnormality in WD and improve both hepatic and neurological symptoms. Studies of the underlying defects in ATP7B and its suspected modifiers ATOX1 and COMMD1 are expected to unravel the diseases genotype–phenotype correlation, and should lead to the design of improved drugs for ameliorating the suffering of patients.

Chromosomal aberrations and sister chromatid exchanges in individuals exposed to arsenic through drinking water in West Bengal, India

Arsenic contamination in groundwater has become a worldwide problem. Currently an unprecedented number of people in West Bengal, India and Bangladesh are exposed to the ubiquitous toxicant via drinking water in exposure levels far exceeding the maximum recommended limit laid down by WHO. This arsenic epidemic has devastated nine districts of West Bengal encompassing an area of 38,865 km(2) leading to various clinical manifestations of chronic arsenicosis. We conducted a human bio-monitoring study using chromosomal aberrations (CA) and sister chromatid exchanges (SCE) as end points to explore the cytogenetic effects of chronic arsenic toxicity in the population of North 24 Parganas, one of the arsenic affected districts in West Bengal. Study participants included 59 individuals residing in this district where the mean level (+/-S.E.) of arsenic in drinking water (microg/l) was 211.70+/-15.28. As age matched controls with similar socio-economic status we selected 36 healthy, asymptomatic individuals residing in two unaffected districts--Midnapur and Howrah where the mean arsenic content of water (microg/l) was 6.35+/-0.45. Exposure was assessed by standardized questionnaires and by detecting the levels of arsenic in drinking water, nails, hair and urine samples. In the exposed group the mean arsenic concentrations in nails (microg/g), hair (microg/g) and urine (microg/l) samples were 9.04+/-0.78, 5.63+/-0.38 and 140.52+/-8.82, respectively, which were significantly high (P<0.01) compared to the corresponding control values of 0.44+/-0.03, 0.30+/-0.02 and 5.91+/-0.49, respectively. Elevated mean values (P<0.01) of the percentage of aberrant cells (8.08%) and SCEs per cell (7.26) were also observed in the exposed individuals in comparison to controls (1.96% and 5.95, respectively). The enhanced rates of CAs and SCEs among the residents of North 24 Parganas are indicative of the cytogenetic damage due to long term exposure to arsenic through consumption of contaminated water.

Cytogenetic damage and genetic variants in the individuals susceptible to arsenic‐induced cancer through drinking water

In West Bengal, India, more than 300,000 arsenic‐exposed people are showing symptoms of arsenic toxicity, which include cancers of skin and different internal organs. Since only 15–20% of the exposed population manifest arsenic‐induced skin lesions, it is thought that genetic variation might play an important role in arsenic toxicity and carcinogenicity. A total of 422 unrelated arsenic‐exposed subjects (244 skin‐symptomatic and 178 asymptomatic) were recruited for this study. Cytogenetic damage, as measured by chromosomal aberrations in lymphocytes and micronuclei formation in oral mucosa cells, urothelial cells and binucleated lymphocytes, was studied in unexposed, skin‐symptomatic and asymptomatic individuals with similar socioeconomic status. Identification of null mutations in GSTT1 and GSTM1 genes were carried out by PCR amplification. GSTP1 SNPs, implicated in susceptibility to various cancers, were assessed by PCR‐RFLP method. Symptomatic individuals had higher level of cytogenetic damage compared to asymptomatic individuals and asymptomatic individuals had significantly higher genotoxicity than unexposed individuals. No difference in allelic variants in GSTT1 and GSTP1 was observed between these 2 groups. Incidence of GSTM1 null gene frequencies was significantly higher in the asymptomatic group. Individuals with GSTM1‐positive (at least one allele) had significantly higher risk of arsenic‐induced skin lesions (odds ratio, 1.73; 95% confidence interval, 1.24–2.22). These results show a protective role of GSTM1 null in arsenic toxicity. This study also indicates that asymptomatic individuals are sub clinically affected and are also significantly susceptible to arsenic‐induced genotoxicity.

Enhanced frequency of micronuclei in individuals exposed to arsenic through drinking water in West Bengal, India.

In West Bengal, India arsenic in ground water has been found to be above the maximum permissible limit in seven districts covering an area of 37,493km2. In the present study, evaluation of the micronuclei (MN) formation in oral mucosa cells, urothelial cells and peripheral blood lymphocytes was carried out in the symptomatic individuals exposed to arsenic through drinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11 microg/l of As in drinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning and residing in two unaffected districts (5.49 microg/l of As) were considered as controls. The exposed and control groups had similar age distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conducted to ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in the study participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair and urine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45 microg/l, 12.58 and 6.97 microg/g, respectively which were significantly high in comparison to corresponding control group values of 4.88 microg/l, 0.51 and 0.34 microg/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oral mucosa, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with the controls (0.77, 0.56 and 0.53/1000 cells, respectively). Thus, the above results indicate that the symptomatic individuals exposed to arsenic through drinking water in this region have significant cytogenetic damage.

The Indian Genome Variation database (IGVdb): A project overview

Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.

Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype–phenotype correlation in Indian patients

Wilson disease (WD) is an autosomal recessive disorder caused by defects in the copper-transporting P-type ATPase gene (ATP7B) resulting in the accumulation of copper in the liver and the brain. We identified prevalent mutations in the ATP7B of Indian WD patients and attempted to correlate those with the disease phenotype. Patients from 62 unrelated families and their first-degree relatives comprising 200 individuals were enrolled in this study. Three dinucleotide repeat markers flanking WD locus and a few intragenic SNPs were used to determine the genotypes and construct haplotypes of the patients. Seven recurring haplotypes accounting for 58% of the total mutant chromosomes were identified, and four underlying defects in the ATP7B representing 37% of WD chromosomes were detected. In addition, five other rare mutations were characterized. Thus a total of nine mutations including five novel changes were identified in the ATP7B of WD patients. Interestingly, homozygotes for different mutations that would be expected to produce similar defective proteins showed significant disparity in terms of organ involvement and severity of the disease. We also observed WD patients with neurological symptoms with little or no manifestation of hepatic pathogenesis. In one WD family, the proband and a sib had remarkably different phenotypes despite sharing the same pair of mutant chromosomes. These findings suggest a potential role for yet unidentified modifying loci for the observed phenotypic heterogeneity among the WD patients.

Myocilin gene implicated in primary congenital glaucoma

Primary congenital glaucoma (PCG) has been associated with CYP1B1 gene (2p21), with a predominantly autosomal recessive mode of inheritance. Our earlier studies attributed CYP1B1 mutations to only 40% of Indian PCG cases. In this study, we included 72 such PCG cases where CYP1B1 mutations were detected in only 12 patients in heterozygous condition, implying involvement of other gene(s). On screening these patients for mutations in myocilin (MYOC), another glaucoma‐associated gene, using denaturing high‐performance liquid chromatography followed by sequencing, we identified a patient who was double heterozygous at CYP1B1 (c.1103G>A; Arg368His) and MYOC (c.144G>T; Gln48His) loci, suggesting a digenic mode of inheritance of PCG. In addition, we identified the same MYOC mutation, implicated for primary open angle glaucoma, in three additional PCG patients who did not harbor any mutation in CYP1B1. These observations suggest a possible role of MYOC in PCG, which might be mediated via digenic interaction with CYP1B1 and/or an yet unidentified locus associated with the disease.

Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India

A study was conducted to explore the effect of arsenic causing conjunctivitis, neuropathy and respiratory illness in individuals, with or without skin lesions, as a result of exposure through drinking water, contaminated with arsenic to similar extent. Exposed study population belongs to the districts of North 24 Parganas and Nadia, West Bengal, India. A total of 725 exposed (373 with skin lesions and 352 without skin lesions) and 389 unexposed individuals were recruited as study participants. Participants were clinically examined and interviewed. Arsenic content in drinking water, urine, nail and hair was estimated. Individuals with skin lesion showed significant retention of arsenic in nail and hair and lower amount of urinary arsenic compared to the group without any skin lesion. Individuals with skin lesion also showed higher risk for conjunctivitis ((odds ratio) OR: 7.33, 95% CI: 5.05–10.59), peripheral neuropathy (OR: 3.95, 95% CI: 2.61–5.93) and respiratory illness (OR: 4.86, 95% CI: 3.16–7.48) compared to the group without any skin lesion. The trend test for OR of the three diseases in three groups was found to be statistically significant. Again, individuals without skin lesion in the exposed group showed higher risk for conjunctivitis (OR: 4.66, 95% CI: 2.45–8.85), neuropathy (OR: 3.99, 95% CI: 1.95–8.09), and respiratory illness (OR: 3.21, 95% CI: 1.65–6.26) when compared to arsenic unexposed individuals. Although individuals with skin lesions were more susceptible to arsenic-induced toxicity, individuals without skin lesions were also subclinically affected and are also susceptible to arsenic-induced toxicity and carcinogenicity when compared to individuals not exposed to arsenic.

Recent advances in molecular genetics of glaucoma.

Glaucoma represents a heterogeneous group of optic neuropathies, with different genetic bases. It can affect all ages generally with a rise in intra-ocular pressure. Three major types of glaucoma have been reported: primary open angle glaucoma (POAG), primary acute closed angle glaucoma (PACG) and primary congenital glaucoma (PCG), as well as a few others associated with developmental abnormalities. In recent years impressive progress has been made in the molecular genetic studies of POAG and PCG. These include the discovery of three genes – Myocilin, Optineurin and CYP1B1 – defects in which results in Mendelian transmission of glaucoma. Identification of single nucleotide polymorphisms in multiple other genes that are associated with glaucoma and alteration of drug sensitivity are enriching our knowledge regarding the complex nature of the disease. This review attempts to present the recent progress made in the molecular genetics of glaucoma.

Toxicogenomics of arsenic: classical ideas and recent advances.

Exposure to arsenic, a toxic metalloid distributed widely in nature, has been known to result in hazardous health outcomes including cancer. Incidence of arsenic toxicity, mostly from usage of underground water, has been reported from different corners of the world spanning more than 21 countries. Recent studies have radically influenced our knowledge on the level of toxicity imparted by different chemical forms of the metalloid. To understand the basic biology for arsenic metabolism different species have been studied at the molecular level, which has unraveled a wealth of information. However, there is no ideal animal model that can be used to understand arsenic toxicity in humans prompting the investigators to undertake the study directly in human cell lines and on affected individuals in areas of exposure. It is interesting to note that only a subset of exposed individuals demonstrate pathological features, i.e. skin lesions sometimes followed by cancer. Also, the extent of affectedness varies remarkably between individuals exposed to similar levels of arsenic. These observations led to investigation of genomic variation in exposed individuals as a potential contributing factor for differential susceptibility. An impressive number of studies have been published on variation in global gene expression and the potential association of candidate genes with arsenic-induced pathology. While there are excellent reviews relating to arsenic metabolism in general, there is no comprehensive review presenting the studies described in humans related to the metalloid toxicity. Our goal in this article has been to present the current state of research on this area to help formulate strategies for future studies.