Genetic architecture of adiposity measures among Asians: Findings from GWAS.

Adiposity has gradually become a global public threat over the years with drastic increase in the attributable deaths and disability adjusted life years (DALYs). Given an increased metabolic risk among Asians as compared to Europeans for any given body mass index (BMI) and considering the differences in genetic architecture between them, the present review aims to summarize the findings from genome-wide scans for various adiposity indices and related anthropometric measures from Asian populations. The search for related studies, published till February 2022, were made on PubMed and GWAS Catalog using search strategy built with relevant keywords joined by Boolean operators. It was recorded that out of a total of 47 identified studies, maximum studies are from Korean population (n = 14), followed by Chinese (n = 7), and Japanese (n = 6). Nearly 200 loci have been identified for BMI, 660 for height, 16 for weight, 28 for circumferences (waist and hip), 32 for ratios (waist hip ratio [WHR] and thoracic hip ratio [THR]), 5 for body fat, 16 for obesity, and 28 for adiposity-related blood markers among Asians. It was observed that though, most of the loci were unique for each trait, there were 3 loci in common to BMI and WHR. Apart from validation of variants identified in European setting, there were many novel loci discovered in Asian populations. Notably, 125 novel loci form Asian studies have been reported for BMI, 47 for height, 5 for waist circumference, and 2 for adiponectin level to the existing knowledge of the genetic framework of adiposity and related measures. It is necessary to examine more advanced adiposity measures, specifically of relevance to abdominal adiposity, a major risk factor for cardiometabolic disorders among Asians. Moreover, in spite of being one continent, there is diversity among different ethnicities across Asia in terms of lifestyle, climate, geography, genetic structure and consequently the phenotypic manifestations. Hence, it is also important to consider ethnic specific studies for identifying and validating reliable genetic variants of adiposity measures among Asians.

Maternal-Periconceptional Vitamin B12 Deficiency in Wistar Rats Leads to Sex-Specific Programming for Cardiometabolic Disease Risk in the Next Generation.

BACKGROUND: Maternal vitamin B12 deficiency plays a vital role in fetal programming, as corroborated by previous studies on murine models and longitudinal human cohorts. OBJECTIVES: This study assessed the effects of diet-induced maternal vitamin B12 deficiency on F1 offspring in terms of cardiometabolic health and normalization of these effects by maternal-periconceptional vitamin B12 supplementation. METHODS: A diet-induced maternal vitamin B12 deficient Wistar rat model was generated in which female rats were either fed a control AIN-76A diet (with 0.01 g/kg vitamin B12) or the same diet with vitamin B12 removed. Females from the vitamin B12-deficient group were mated with males on the control diet. A subset of vitamin B12-deficient females was repleted with vitamin B12 on day 1 of conception. The offspring in the F1 generation were assessed for changes in body composition, plasma biochemistry, and molecular changes in the liver. A multiomics approach was used to obtain a mechanistic insight into the changes in the offspring liver. RESULTS: We showed that a 36% reduction in plasma vitamin B12 levels during pregnancy in F0 females can lead to continued vitamin B12 deficiency (60%-70% compared with control) in the F1 offspring and program them for cardiometabolic adversities. These adversities, such as high triglycerides and low high-density lipoprotein cholesterol, were seen only among F1 males but not females. DNA methylome analysis of the liver of F1 3-mo-old offspring highlights sexual dimorphism in the alteration of methylation status of genes critical to signaling processes. Proteomics and targeted metabolomics analysis confirm that sex-specific alterations occur through modulations in PPAR signaling and steroid hormone biosynthesis pathway. Repletion of deficient mothers with vitamin B12 at conception normalizes most of the molecular and biochemical changes. CONCLUSIONS: Maternal vitamin B12 deficiency has a programming effect on the next generation and increases the risk for cardiometabolic syndrome in a sex-specific manner. Normalization of the molecular risk markers on vitamin B12 supplementation indicates a causal role.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Protocol for a cluster randomised trial evaluating a multifaceted intervention starting preconceptionally-Early Interventions to Support Trajectories for Healthy Life in India (EINSTEIN): a Healthy Life Trajectories Initiative (HeLTI) Study.

INTRODUCTION: The Healthy Life Trajectories Initiative is an international consortium comprising four harmonised but independently powered trials to evaluate whether an integrated intervention starting preconceptionally will reduce non-communicable disease risk in their children. This paper describes the protocol of the India study. METHODS AND ANALYSIS: The study set in rural Mysore will recruit ~6000 married women over the age of 18 years. The village-based cluster randomised design has three arms (preconception, pregnancy and control; 35 villages per arm). The longitudinal multifaceted intervention package will be delivered by community health workers and comprise: (1) measures to optimise nutrition; (2) a group parenting programme integrated with cognitive-behavioral therapy; (3) a lifestyle behaviour change intervention to support women to achieve a diverse diet, exclusive breast feeding for the first 6 months, timely introduction of diverse and nutritious infant weaning foods, and adopt appropriate hygiene measures; and (4) the reduction of environmental pollution focusing on indoor air pollution and toxin avoidance.The primary outcome is adiposity in children at age 5 years, measured by fat mass index. We will report on a host of intermediate and process outcomes. We will collect a range of biospecimens including blood, urine, stool and saliva from the mothers, as well as umbilical cord blood, placenta and specimens from the offspring.An intention-to-treat analysis will be adopted to assess the effect of interventions on outcomes. We will also undertake process and economic evaluations to determine scalability and public health translation. ETHICS AND DISSEMINATION: The study has been approved by the institutional ethics committee of the lead institute. Findings will be published in peer-reviewed journals. We will interact with policy makers at local, national and international agencies to enable translation. We will also share the findings with the participants and local community through community meetings, newsletters and local radio. TRIAL REGISTRATION NUMBER: ISRCTN20161479, CTRI/2020/12/030134; Pre-results.

Maternal vitamin B12 deficiency in rats alters DNA methylation in metabolically important genes in their offspring.

Vitamin B12 deficiency is a critical problem worldwide and peri-conceptional deficiency of this vitamin is associated with the risk of complex cardio-metabolic diseases. Nutritional perturbations during these stages of development may lead to changes in the fetal epigenome. Using Wistar rat model system, we have earlier shown that low maternal B12 levels are associated with low birth weight, adiposity, insulin resistance, and increased triglyceride levels in the offspring, which might predispose them to the risk of cardio-metabolic diseases in adulthood. In this study, we have investigated the effects of maternal B12 deficiency on genome-wide DNA methylation profile of the offspring and the effect of rehabilitation of mothers with B12 at conception. We have performed methylated DNA immunoprecipitation sequencing of liver from pups in four groups of Wistar rats: Control (C), B12-restricted (B12R), B12-rehabilitated at conception (B12RC), and B12-rehabilitated at parturition (B12RP). We have analyzed differentially methylated signatures between the three groups as compared to controls. We have identified a total of 214 hypermethylated and 142 hypomethylated regions in the 10 kb upstream region of transcription start site in pups of B12-deficient mothers, which are enriched in genes involved in fatty acid metabolism and mitochondrial transport/metabolism. B12 rehabilitation at conception and parturition is responsible for reversal of methylation status of many of these regions to control levels suggesting a causal association with metabolic phenotypes. Thus, maternal B12 restriction alters DNA methylation of genes involved in important metabolic processes and influences the offspring phenotype, which is reversed by B12 rehabilitation of mothers at conception.

Causal relationships between lipid and glycemic levels in an Indian population: A bidirectional Mendelian randomization approach.

BACKGROUND: Dyslipidemia and abnormal glycemic traits are leading causes of morbidity and mortality. Although the association between the two traits is well established, there still exists a gap in the evidence for the direction of causality. OBJECTIVE: This study aimed to examine the direction of the causal relationship between lipids and glycemic traits in an Indian population using bidirectional Mendelian randomization (BMR). METHODS: The BMR analysis was conducted on 4900 individuals (2450 sib-pairs) from the Indian Migration Study. Instrument variables were generated for each lipid and glycemic trait (fasting insulin, fasting glucose, HOMA-IR, HOMA-ß, LDL-cholesterol, HDL-cholesterol, total cholesterol and triglycerides) to examine the causal relationship by applying two-stage least squares (2SLS) regression in both directions. RESULTS: Lipid and glycemic traits were found to be associated observationally, however, results from 2SLS showed that only triglycerides, defined by weighted genetic risk score (wGRS) of 3 SNPs (rs662799 at APOAV, rs780094 at GCKR and rs4420638 at APOE/C1/C4), were observed to be causally effecting 1.15% variation in HOMA-IR (SE = 0.22, P = 0.010), 1.53% in HOMA- ß (SE = 0.21, P = 0.001) and 1.18% in fasting insulin (SE = 0.23, P = 0.009). No evidence for a causal effect was observed in the reverse direction or between any other lipid and glycemic traits. CONCLUSION: The study findings suggest that triglycerides may causally impact various glycemic traits. However, the findings need to be replicated in larger studies.

Association Analysis of PRSS1-PRSS2 and CLDN2-MORC4 Variants in Nonalcoholic Chronic Pancreatitis Using Tropical Calcific Pancreatitis as Model.

OBJECTIVE: Association of PRSS1-PRSS2 (rs10273639) and CLDN2-MORC4 (rs12688220 and rs7057398) variants with alcohol-related chronic pancreatitis (CP) is established but with nonalcoholic CP is unclear. We addressed this inconsistency using tropical calcific pancreatitis (TCP) as model. METHODS: We sequenced 5'-UTR of PRSS1 and genotyped CLDN2-MORC4 variants in 555 patients with TCP and 801 controls and performed association analysis. Gene-gene interaction between PRSS1 and CLDN2-MORC4 variants and with p.Asn34Ser SPINK1 and p.Leu26Val CTSB was also evaluated. RESULTS: We observed significant association of rs10273639/rs4726576 in PRSS1-PRSS2 (odds ratio [OR] = 0.72; P = 3.50 × 10) and CLDN2-MORC4 variants, rs12688220 (OR = 1.54; P = 1.22 × 10) and rs7057398 (OR = 1.50; P = 1.22 × 10) with TCP. Patients carrying p.Asn34Ser SPINK1 were significantly younger than those with rs4726576 risk genotype (30.0 vs 38.0 years; P = 0.015) and those carrying both were even younger (22.0 years; P = 0.001). Presence of risk allele at rs12688220 in patients carrying p.Asn34Ser SPINK1 delayed the age of onset (32.0 vs 24.0 years; P = 0.013). CONCLUSIONS: Our study establishes strong association of PRSS1-PRSS2 and CLDN2-MORC4 variants with TCP and thus with nonalcoholic CP. These variants independently interact with p.Asn34Ser SPINK1 and influence the age of onset in TCP. However, latter results need to be replicated in other cohorts.

Intrauterine Programming of Diabetes and Adiposity.

The prevalence of diabetes and adiposity has increased at an alarming rate and together they contribute to the rise in morbidity and mortality worldwide. Genetic studies till date have succeeded in explaining only a proportion of heritability, while a major component remains unexplained. Early life determinants of future risk of these diseases are likely contributors to the missing heritability and thus have a significant potential in disease prevention. Epidemiological and animal studies show the importance of intrauterine and early postnatal environment in programming of the fetus to adverse metabolic outcomes and support the notion of Developmental Origins of Health and Disease (DOHaD). Emerging evidence highlights the role of epigenetic mechanisms in mediating effects of environmental exposures, which in certain instances may exhibit intergenerational transmission even in the absence of exposure. In this article, we will discuss the complexity of diabetes and increased adiposity and mechanisms of programming of these adverse metabolic conditions.

Maternal micronutrient deficiency leads to alteration in the kidney proteome in rat pups.

Maternal nutritional deficiency significantly perturbs the offspring's physiology predisposing them to metabolic diseases during adulthood. Vitamin B12 and folate are two such micronutrients, whose deficiency leads to elevated homocysteine levels. We earlier generated B12 and/or folate deficient rat models and using high-throughput proteomic approach, showed that maternal vitamin B12 deficiency modulates carbohydrate and lipid metabolism in the liver of pups through regulation of PPAR signaling pathway. In this study, using similar approach, we identified 26 differentially expressed proteins in the kidney of pups born to mothers fed with vitamin B12 deficient diet while only four proteins were identified in the folate deficient group. Importantly, proteins like calreticulin, cofilin 1 and nucleoside diphosphate kinase B that are involved in the functioning of the kidney were upregulated in B12 deficient group. Our results hint towards a larger effect of vitamin B12 deficiency compared to that of folate presumably due to greater elevation of homocysteine in vitamin B12 deficient group. In view of widespread vitamin B12 and folate deficiency and its association with several diseases like anemia, cardiovascular and renal diseases, our results may have large implications for kidney diseases in populations deficient in vitamin B12 especially in vegetarians and the elderly people.This article is part of a Special Issue entitled: Proteomics in India.

Chronic maternal vitamin B12 restriction induced changes in body composition & glucose metabolism in the Wistar rat offspring are partly correctable by rehabilitation.

Maternal under-nutrition increases the risk of developing metabolic diseases. We studied the effects of chronic maternal dietary vitamin B12 restriction on lean body mass (LBM), fat free mass (FFM), muscle function, glucose tolerance and metabolism in Wistar rat offspring. Prevention/reversibility of changes by rehabilitating restricted mothers from conception or parturition and their offspring from weaning was assessed. Female weaning Wistar rats (n = 30) were fed ad libitum for 12 weeks, a control diet (n = 6) or the same with 40% restriction of vitamin B12 (B12R) (n = 24); after confirming deficiency, were mated with control males. Six each of pregnant B12R dams were rehabilitated from conception and parturition and their offspring weaned to control diet. While offspring of six B12R dams were weaned to control diet, those of the remaining six B12R dams continued on B12R diet. Biochemical parameters and body composition were determined in dams before mating and in male offspring at 3, 6, 9 and 12 months of their age. Dietary vitamin B12 restriction increased body weight but decreased LBM% and FFM% but not the percent of tissue associated fat (TAF%) in dams. Maternal B12R decreased LBM% and FFM% in the male offspring, but their TAF%, basal and insulin stimulated glucose uptake by diaphragm were unaltered. At 12 months age, B12R offspring had higher (than controls) fasting plasma glucose, insulin, HOMA-IR and impaired glucose tolerance. Their hepatic gluconeogenic enzyme activities were increased. B12R offspring had increased oxidative stress and decreased antioxidant status. Changes in body composition, glucose metabolism and stress were reversed by rehabilitating B12R dams from conception, whereas rehabilitation from parturition and weaning corrected them partially, highlighting the importance of vitamin B12 during pregnancy and lactation on growth, muscle development, glucose tolerance and metabolism in the offspring.

Association of common genetic variants with lipid traits in the Indian population.

Genome-wide association studies (GWAS) have been instrumental in identifying novel genetic variants associated with altered plasma lipid levels. However, these quantitative trait loci have not been tested in the Indian population, where there is a poorly understood and growing burden of cardiometabolic disorders. We present the association of six single nucleotide polymorphisms in 1671 sib pairs (3342 subjects) with four lipid traits: total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). We also investigated the interaction effects of gender, location, fat intake and physical activity. Each copy of the risk allele of rs964184 at APOA1 was associated with 1.06 mmol/l increase in triglycerides (SE = 0.049; p = 0.006), rs3764261 at CETP with 1.02 mmol/l increase in both total cholesterol (SE = 0.042; p = 0.017) and HDL-C (SE = 0.041; p = 0.008), rs646776 at CELSR2-PSRC1-SORT1 with 0.96 mmol/l decrease in cholesterol (SE = 0.043; p = 0.0003) and 0.15 mmol/l decrease in LDL-C levels (SE = 0.043; p = 0.0003) and rs2954029 at TRIB1 with 1.02 mmol/l increase in HDL-C (SE = 0.039; p = 0.047). A combined risk score of APOA1 and CETP loci predicted an increase of 1.25 mmol/l in HDL-C level (SE = 0.312; p = 0.0007). Urban location and sex had strong interaction effects on the genetic association of most of the studied loci with lipid traits. To conclude, we validated four genetic variants (identified by GWAS in western populations) associated with lipid traits in the Indian population. The interaction effects found here may explain the sex-specific differences in lipid levels and their heritability. Urbanization appears to influence the nature of the association with GWAS lipid loci in this population. However, these findings will require replication in other Indian populations.

Variants in CPA1 are strongly associated with early onset chronic pancreatitis.

Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1, encoding carboxypeptidase A1, in subjects with nonalcoholic chronic pancreatitis (cases) and controls in a German discovery set and three replication sets. Functionally impaired variants were present in 29/944 (3.1%) German cases and 5/3,938 (0.1%) controls (odds ratio (OR) = 24.9, P = 1.5 × 10(-16)). The association was strongest in subjects aged ≤ 10 years (9.7%; OR = 84.0, P = 4.1 × 10(-24)). In the replication sets, defective CPA1 variants were present in 8/600 (1.3%) cases and 9/2,432 (0.4%) controls from Europe (P = 0.01), 5/230 (2.2%) cases and 0/264 controls from India (P = 0.02) and 5/247 (2.0%) cases and 0/341 controls from Japan (P = 0.013). The mechanism by which CPA1 variants confer increased pancreatitis risk may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity, as is seen with other genetic risk factors for this disease.

PPAR signaling pathway is a key modulator of liver proteome in pups born to vitamin B(12) deficient rats.

Maternal nutritional deficiency in-utero is known to predict risk of complex disorders like cardiovascular disease, diabetes and many neurological disorders in the offspring and vitamin B12 is one such critical micronutrient. Here we performed 2D-DIGE followed by MALDI TOF/TOF analysis to identify proteins that are differentially expressed in liver of pups born to mothers fed vitamin B12 deficient diet vis-à-vis control diet. To further establish causality, we analyzed the effect of B12 rehabilitation at parturition on the protein levels and the phenotype in pups. We identified 38 differentially expressed proteins that were enriched in pathways involved in the regulation of amino acid, lipid and carbohydrate metabolism. Further, three enzymes in the ß-oxidation pathway (hydroxyacyl-coenzyme A dehydrogenase, medium-chain specific acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase) were down-regulated in pups born to mothers fed vitamin B12 deficient diet. We observed age-dependent differential expression of peroxisome proliferator activated-receptor (PPAR) α and γ in the deficient pups. Interestingly, expression of 27 proteins that were differentially expressed was restored to the control levels after rehabilitation of female rats with vitamin B12 from parturition. Our study thus provides the first evidence that maternal vitamin B12 deficiency influences lipid and other micronutrient metabolism in pups through regulation of PPAR signaling pathway. BIOLOGICAL SIGNIFICANCE: Maternal vitamin B12 deficiency has been shown to predict the onset of complex disorders like atherosclerosis, type II diabetes etc. in the next generation during their adulthood. We have shown earlier that pups born to female rats fed with vitamin B12 deficient diet were obese and developed high levels of other intermediate traits such as triglycerides, cholesterol etc. that are related to the risk of diabetes and cardiovascular disorders. In this piece of work using differential proteomic approach we have identified the altered metabolic processes in the liver of vitamin B12 deficient pups. We have also documented that the proteins involved in ß-oxidation pathway are down-regulated. Further, differential expression of PPARα and PPARγ was evidently documented as the master regulator for the alteration of lipid, amino acid and carbohydrate metabolism during maternal vitamin B12 deficiency.

Association study of 25 type 2 diabetes related Loci with measures of obesity in Indian sib pairs.

Obesity is an established risk factor for type 2 diabetes (T2D) and they are metabolically related through the mechanism of insulin resistance. In order to explore how common genetic variants associated with T2D correlate with body mass index (BMI), we examined the influence of 25 T2D associated loci on obesity risk. We used 5056 individuals (2528 sib-pairs) recruited in Indian Migration Study and conducted within sib-pair analysis for six obesity phenotypes. We found associations of variants in CXCR4 (rs932206) and HHEX (rs5015480) with higher body mass index (BMI) (ß=0.13, p=0.001) and (ß=0.09, p=0.002), respectively and weight (ß=0.13, p=0.001) and (ß=0.09, p=0.001), respectively. CXCR4 variant was also strongly associated with body fat (ß=0.10, p=0.0004). In addition, we demonstrated associations of CXCR4 and HHEX with overweight/obesity (OR=1.6, p=0.003) and (OR=1.4, p=0.002), respectively, in 1333 sib-pairs (2666 individuals). We observed marginal evidence of associations between variants at six loci (TCF7L2, NGN3, FOXA2, LOC646279, FLJ39370 and THADA) and waist hip ratio (WHR), BMI and/or overweight which needs to be validated in larger set of samples. All the above findings were independent of daily energy consumption and physical activity level. The risk score estimates based on eight significant loci (including nominal associations) showed associations with WHR and body fat which were independent of BMI. In summary, we establish the role of T2D associated loci in influencing the measures of obesity in Indian population, suggesting common underlying pathophysiology across populations.

Comprehensive functional analysis of chymotrypsin C (CTRC) variants reveals distinct loss-of-function mechanisms associated with pancreatitis risk.

OBJECTIVE: The digestive enzyme chymotrypsin C (CTRC) protects against pancreatitis by promoting degradation of trypsinogen, thereby curtailing potentially harmful trypsinogen activation. Loss-of-function variants in CTRC increase the risk for chronic pancreatitis. The aim of the present study was to perform comprehensive functional analysis of all missense CTRC variants identified to date. DESIGN: We investigated secretion, activity and degradation of 27 published and five novel CTRC mutants. We also assessed the effect of five mutants on endoplasmic reticulum (ER) stress. RESULTS: None of the mutants exhibited a gain of function, such as increased secretion or activity. By contrast, 11 mutants showed marked loss of function, three mutants had moderate functional defects, whereas 18 mutants were functionally similar to wild-type CTRC. The functional deficiencies observed were diminished secretion, impaired catalytic activity and degradation by trypsin. Mutants with a secretion defect caused ER stress that was proportional to the loss in secretion. ER stress was not associated with loss-of-function phenotypes related to catalytic defect or proteolytic instability. CONCLUSIONS: Pathogenic CTRC variants cause loss of function by three distinct but mutually non-exclusive mechanisms that affect secretion, activity and proteolytic stability. ER stress may be induced by a subset of CTRC mutants, but does not represent a common pathological mechanism of CTRC variants. This phenotypic dataset should aid in the classification of the clinical relevance of CTRC variants identified in patients with chronic pancreatitis.

Comprehensive screening of chymotrypsin C (CTRC) gene in tropical calcific pancreatitis identifies novel variants.

OBJECTIVE: In a previous study, the authors have shown that rather than variants in trypsinogen gene(s), mutations in pancreatic secretory trypsin inhibitor (encoded by SPINK1) and cathepsin B (CTSB) are associated with tropical calcific pancreatitis (TCP). Recently, chymotrypsin C (CTRC) variants that diminish its activity or secretion were found to predict susceptibility to chronic pancreatitis (CP). The authors analysed CTRC variants in a large, ethnically matched case-control TCP cohort. DESIGN: The authors sequenced all eight exons and flanking regions in CTRC in 584 CP patients (497 TCP, 87 idiopathic CP) and 598 normal subjects and analysed the significance of association using χ(2) test. The authors also investigated interaction of CTRC variants with p.N34S SPINK1 and p.L26V CTSB mutations. RESULTS: The authors identified 14 variants in CTRC, of which non-synonymous variants were detected in 71/584 CP patients (12.2%) and 22/598 controls (3.7%; OR 3.62, 95% CI 2.21 to 5.93; p=6.2 × 10(-8)). Rather than the commonly reported p.K247_R254del variant in Caucasians, p.V235I was the most common mutation in Indian CP patients (28/575 (4.9%); OR 7.60, 95% CI 2.52 to 25.71; p=1.01 × 10(-5)). Another pathogenic variant, p.A73T was identified in 3.1% (18/584) patients compared with 0.3% (2/598) in controls (OR=9.48, 95% CI 2.19 to 41.03, p=2.5 × 10(-4)). The authors also observed significant association for the synonymous variant c.180C>T (p.(=)) with CP (OR 2.71, 95% CI 1.79 to 4.12, p=5.3 × 10(-7)). Two novel nonsense mutations, p.G242AfsX9 and p.W113X were also identified exclusively in CP patients. No interaction between CTRC variants and p.N34S SPINK1 or p.L26V CTSB mutations was observed. CONCLUSION: This study on a large cohort of TCP patients provides evidence of allelic heterogeneity and confirms that CTRC variants play a significant role in its pathogenesis.

Maternal dietary folate and/or vitamin B12 restrictions alter body composition (adiposity) and lipid metabolism in Wistar rat offspring.

Maternal vitamin deficiencies are associated with low birth weight and increased perinatal morbidity and mortality. We hypothesize that maternal folate and/or vitamin B(12) restrictions alter body composition and fat metabolism in the offspring. Female weaning Wistar rats received ad libitum for 12 weeks a control diet (American Institute of Nutrition-76A) or the same with restriction of folate, vitamin B(12) or both (dual deficient) and, after confirming vitamin deficiency, were mated with control males. The pregnant/lactating mothers and their offspring received their respective diets throughout. Biochemical and body composition parameters were determined in mothers before mating and in offspring at 3, 6, 9 and 12 months of age. Vitamin restriction increased body weight and fat and altered lipid profile in female Wistar rats, albeit differences were significant with only B(12) restriction. Offspring born to vitamin-B(12)-restricted dams had lower birth weight, while offspring of all vitamin-restricted dams weighed higher at/from weaning. They had higher body fat (specially visceral fat) from 3 months and were dyslipidemic at 12 months, when they had high circulating and adipose tissue levels of tumor necrosis factor α, leptin and interleukin 6 and low levels of adiponectin and interleukin 1ß. Vitamin-restricted offspring had higher activities of hepatic fatty acid synthase and acetyl-CoA-carboxylase and higher plasma cortisol levels. In conclusion, maternal and peri-/postnatal folate and/or vitamin B(12) restriction increased visceral adiposity (due to increased corticosteroid stress), altered lipid metabolism in rat offspring perhaps by modulating adipocyte function and may thus predispose them to high morbidity later.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy.

PURPOSE: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. METHODS: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. RESULTS: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. CONCLUSIONS: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

High resolution methylome map of rat indicates role of intragenic DNA methylation in identification of coding region.

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability.