The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (K_D of 0.15 ± 0.06 nM) and its N34S variant (K_D of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.     

Pathways to injury in chronic pancreatitis: decoding the role of the high-risk SPINK1 N34S haplotype using meta-analysis

Background

The complex interactions between recurrent trypsin-mediated pancreatic injury, alcohol-associated pancreatic injury and SPINK1 polymorphisms in chronic pancreatitis (CP) are undefined. We hypothesize that CP occurs as a result of multiple pathological mechanisms (pathways) that are initiated by different metabolic or environmental factors (etiologies) and may be influenced differentially by downstream genetic risk factors. We tested this hypothesis by evaluating the differences in effect size of the high risk SPINK1 N34S haplotype on CP from multiple etiologies after combining clinical reports of SPINK1 N34S frequency using meta-analysis.

Methods and Findings

The Pubmed and the Embase databases were reviewed. We studied 24 reports of SPINK1 N34S in CP (2,421 cases, 4,857 controls) using reported etiological factors as surrogates for pathways and multiple meta-analyses to determine the differential effects of SPINK1 N34S between alcoholic and non-alcoholic etiologies. Using estimates of between-study heterogeneity, we sub-classified our 24 studies into four specific clusters. We found that SPINK1 N34S is strongly associated with CP overall (OR 11.00; 95% CI: 7.59–15.93), but the effect of SPINK1 N34S in alcoholic CP (OR 4.98, 95% CI: 3.16–7.85) was significantly smaller than in idiopathic CP (OR 14.97, 95% C.I. = 9.09–24.67) or tropical CP (OR 19.15, 95% C.I. = 8.83–41.56). Studies analyzing familial CP showed very high heterogeneity suggestive of a complex etiology with an I² = 80.95%.

Conclusion

The small effect of SPINK1 N34S in alcoholic subjects suggests that CP is driven through a different pathway that is largely trypsin-independent. The results also suggest that large effect sizes of SPINK1 N34S in small candidate gene studies in CP may be related to a mixture of multiple etiologic pathways leading to the same clinical endpoint.     

Spectrum and distribution of CFTR gene mutations in asthma and chronic pancreatitis cases of North Indian population

Background

Cystic fibrosis transmembrane conductance regulator (CFTR) gene accounts for an autosomal recessive condition called cystic fibrosis (CF). In the Indian subcontinent, CF and its related diseases are under-diagnosed by the medical community due to poor knowledge of the disease and its confounding diagnosis, and also due to poor medical facilities available for these patients, thus causing an increased infant mortality rate with a low life expectancy in general. The aim of the study was to document the spectrum and distribution of CFTR mutations in controls, asthma and chronic pancreatitis cases of North India.

Methods

A total of 800 subjects including 400 controls, 250 asthma cases and150 chronic pancreatitis cases were analyzed for 6 mutations (F508del, G542X, G551D, R117H, W1282X, and S549N) and IVS8 Tn polymorphism.

Results

Out of 800 subjects, 18% [asthma — 24% (n = 250), CP — 29.33% (n = 150) cases and controls — 9.3% (n = 400)] were positive for heterozygous mutation, 0.8% of the (n = 250) asthmatic cases (n = 250) were homozygous for IVS8 T5 polymorphism while no subjects were found positive for W1282X mutation. T5 polymorphism was more common in asthmatic cases while F508del mutation in chronic pancreatitis cases. The carrier frequency of F508del, G542X, G551D, R117H, S549N and T5 was 0.015, 0.025, 0.02, 0.005, 0.005, and 0.022 respectively. The cumulative carrier frequency was 0.093.

Conclusion

CFTR mutations were underestimated in Indian population. The present study will serve in establishment of genetic screening and prenatal setup for Indian population.     

Erythrocyte insulin binding abnormalities in fibro calculous pancreatic diabetes

Insulin binding to erythrocyte insulin receptors was studied in 17 patients (13 men and 4 women) with fibrocalculous pancreatic diabetes mellitus (FCPD) and compared with that of 14 newly diagnosed NIDDM patients matched for age, sex and severity of hyperglycemia, and 14 age and sex-matched non-diabetic control subjects. In the uncompensated diabetic state, mean (+/- S.D.) specific binding of insulin was lower in both FCPD and NIDDM patients, compared with non-diabetic controls (P less than 0.001). Control of diabetes with short term therapy (2-6 weeks) resulted in a significant improvement in the mean specific insulin binding in both FCPD and NIDDM patients (P less than 0.001) due to increased binding affinity in the former, and increased affinity and the number of binding sites in the latter. As compared to short term therapy, chronic therapy (5-8 months) in FCPD patients resulted in a marginal decrease in specific insulin binding. However, this was still significantly higher than the basal value (P less than 0.05). FCPD patients had an initial low mean basal plasma IRI and a much lower mean stimulated IRI response as compared to NIDDM and non-diabetic controls.     

Quantitative Trait Locus Analysis for Chronic Pancreatitis and Diabetes Mellitus in the WBN/Kob Rat

A causative gene mutation is still undefined in approximately half of patients with hereditary pancreatitis, and no genetic factor has been identified in most patients with sporadic chronic pancreatitis. To identify a pancreatitis-associated gene, we performed a quantitative trait locus (QTL) analysis for the traits of chronic pancreatitis and diabetes mellitus in WBN/Kob rats. We identified two highly significant QTLs for chronic pancreatitis and/or hyperinsulinemia on chromosomes 7 and X. These QTLs were located on completely different chromosomal regions from those of causative genes that have been reported for human chronic pancreatitis: PRSS1, CFTR, and SPINK1. For these QTLs, prevalences of the WBN/Kob allele significantly increased in the rats with chronic pancreatitis. These findings indicate that chronic pancreatitis in WBN/Kob rats is controlled by multiple genes, and a genetic analysis in WBN/Kob rats might be useful for gene targeting for human chronic pancreatitis.     

Alstrom syndrome (OMIM 203800): a case report and literature review

Hereditary chronic pancreatitis (HCP) is a very rare form of early onset chronic pancreatitis. With the exception of the young age at diagnosis and a slower progression, the clinical course, morphological features and laboratory findings of HCP do not differ from those of patients with alcoholic chronic pancreatitis. As well, diagnostic criteria and treatment of HCP resemble that of chronic pancreatitis of other causes. The clinical presentation is highly variable and includes chronic abdominal pain, impairment of endocrine and exocrine pancreatic function, nausea and vomiting, maldigestion, diabetes, pseudocysts, bile duct and duodenal obstruction, and rarely pancreatic cancer. Fortunately, most patients have a mild disease. Mutations in the PRSS1 gene, encoding cationic trypsinogen, play a causative role in chronic pancreatitis. It has been shown that the PRSS1 mutations increase autocatalytic conversion of trypsinogen to active trypsin, and thus probably cause premature, intrapancreatic trypsinogen activation disturbing the intrapancreatic balance of proteases and their inhibitors. Other genes, such as the anionic trypsinogen (PRSS2), the serine protease inhibitor, Kazal type 1 (SPINK1) and the cystic fibrosis transmembrane conductance regulator (CFTR) have been found to be associated with chronic pancreatitis (idiopathic and hereditary) as well. Genetic testing should only be performed in carefully selected patients by direct DNA sequencing and antenatal diagnosis should not be encouraged. Treatment focuses on enzyme and nutritional supplementation, pain management, pancreatic diabetes, and local organ complications, such as pseudocysts, bile duct or duodenal obstruction. The disease course and prognosis of patients with HCP is unpredictable. Pancreatic cancer risk is elevated. Therefore, HCP patients should strongly avoid environmental risk factors for pancreatic cancer.     

Genetic variants of cytoplasmic malate dehydrogenase (MDH:EC:1.1.1.37) in populations in England and the Indian subcontinent

Summary A new variant malate dehydrogenase is described, designated S-MDH^Ind to indicate its discovery in the Indian subcontinent pending full comparison with all other variants. It occurred during a survey of the incidence of variant S-MDH phenotypes in 4149 subjects in north-east England and 1494 subjects from several populations in the Indian region. The variants previously thought to be restricted to the New Guinea region and to African populations occurred in three English subjects in northeast England. The incidence of variant S-MDH phenotypes in other populations is summarised.     

Molecular dynamics simulations reveal structural instability of human trypsin inhibitor upon D50E and Y54H mutations

Serine protease inhibitor Kazal type 1 (SPINK1) plays an important role in protecting the pancreas against premature trypsinogen activation that causes pancreatitis. Various mutations in the SPINK1 gene were shown to be associated with patients with pancreatitis. Recent transfection studies identified intracellular folding defects, probably caused by mutation induced misfolding of D50E and Y54H mutations, as a common mechanism that reduces SPINK1 secretion and as a possible novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. Using molecular dynamics, we investigated the effects of D50E and Y54H mutations on SPINK1 dynamics and conformation at 300 K. We found that the structures of D50E and Y54H mutants were less stable than and were distorted from those of the wild type, as indicated by the RMSD plots, RMSF plots and DSSP series. Specifically, unwinding of the top of helices (the main secondary structures) and the distortion of the loops above the helices were observed. It may be possible that this distorted protein structure may be recognized as “non-native” by members of the chaperone family; it may be further retained and targeted for degradation, leading to SPINK1 secretion reduction and subsequently pancreatitis in patients as Király et al. (Gut 56:1433, 2007) proposed.     

A Conservative Assessment of the Major Genetic Causes of Idiopathic Chronic Pancreatitis: Data from a Comprehensive Analysis of PRSS1, SPINK1, CTRC and CFTR Genes in 253 Young French Patients

Idiopathic chronic pancreatitis (ICP) has traditionally been defined as chronic pancreatitis in the absence of any obvious precipitating factors (e.g. alcohol abuse) and family history of the disease. Studies over the past 15 years have revealed that ICP has a highly complex genetic architecture involving multiple gene loci. Here, we have attempted to provide a conservative assessment of the major genetic causes of ICP in a sample of 253 young French ICP patients. For the first time, conventional types of mutation (comprising coding sequence variants and variants at intron/exon boundaries) and gross genomic rearrangements were screened for in all four major pancreatitis genes, PRSS1, SPINK1, CTRC and CFTR. For the purposes of the study, synonymous, intronic and 5''- or 3''-untranslated region variants were excluded from the analysis except where there was persuasive evidence of functional consequences. The remaining sequence variants/genotypes were classified into causative, contributory or neutral categories by consideration of (i) their allele frequencies in patient and normal control populations, (ii) their presumed or experimentally confirmed functional effects, (iii) the relative importance of their associated genes in the pathogenesis of chronic pancreatitis and (iv) gene-gene interactions wherever applicable. Adoption of this strategy allowed us to assess the pathogenic relevance of specific variants/genotypes to their respective carriers to an unprecedented degree. The genetic cause of ICP could be assigned in 23.7% of individuals in the study group. A strong genetic susceptibility factor was also present in an additional 24.5% of cases. Taken together, up to 48.2% of the studied ICP patients were found to display evidence of a genetic basis for their pancreatitis. Whereas these particular proportions may not be extrapolable to all ICP patients, the approach employed should serve as a useful framework for acquiring a better understanding of the role of genetic factors in causing this oligogenic disease.     

Gc subtypes in Northern Indians

Isoelectric focusing was used to determine the frequencies of the Gc subtypes in a population sample from The North Indian subcontinent (now living in Birmingham, UK). The gene frequencies observed were as follows: Gc1F = 0.191, Gc1S = 0.519 and Gc2 = 0.290.243 individuals were typed and no variant alleles were detected.     

Genomewide scan for nonsyndromic cleft lip and palate in multigenerational Indian families reveals significant evidence of linkage at 13q33.1-34

Nonsyndromic cleft lip with or without cleft palate (CL-P) is a common congenital anomaly with incidence ranging from 1 in 300 to 1 in 2,500 live births. We analyzed two Indian pedigrees (UR017 and UR019) with isolated, nonsyndromic CL-P, in which the anomaly segregates as an autosomal dominant trait. The phenotype was variable, ranging from unilateral to bilateral CL-P. A genomewide linkage scan that used approximately 10,000 SNPs was performed. Nonparametric linkage (NPL) analysis identified 11 genomic regions (NPL>3.5; P<.005) that could potentially harbor CL-P susceptibility variations. Among those, the most significant evidence was for chromosome 13q33.1-34 at marker rs1830756 (NPL=5.57; P=.00024). This was also supported by parametric linkage; MOD score (LOD scores maximized over genetic model parameters) analysis favored an autosomal dominant model. The maximum LOD score was 4.45, and heterogeneity LOD was 4.45 (alpha =100%). Haplotype analysis with informative crossovers enabled the mapping of the CL-P locus to a region of approximately 20.17 cM (7.42 Mb) between SNPs rs951095 and rs726455. Thus, we have identified a novel genomic region on 13q33.1-34 that harbors a high-risk variant for CL-P in these Indian families.     

Mutational Analysis of ATP8B1 in Patients with Chronic Pancreatitis

Background

Mutations in genes encoding cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency.

Methods

We analyzed all 27 ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls.

Results

In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups.

Conclusions

We did not find an association between heterozygous ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.     

Frequency of Strongyloides stercoralis infection in alcoholics

Several studies have shown that chronic alcoholics have increased susceptibility to infections due to higher exposure to infectious agents as well as breakdown in their immune defenses. As Strongyloides stercoralis infection is usually more relevant in immunocompromised patients, the aim of this study was to evaluate the frequency of S. stercoralis infection in alcoholics. Thus, coproparasitological examination was carried out in 145 subjects, from which 45 were chronic alcoholics (mean age of 45.7 +/- 11.0 years), 10 were nonalcoholic cirrhotic patients (mean age of 50.2 +/- 13.1 years), and 90 were asymptomatic nonalcoholic subjects (mean age of 46.7 +/- 10.1 years), which served as controls. From the alcoholics, 9 had hepatic cirrhosis, 9 had chronic pancreatitis and 27 had neither cirrhosis nor pancreatitis. For the diagnosis of strongyloidiasis, the Baermann-Moraes and Lutz methods were used in three fecal samples from each subject. Samples were collected at alternated days, and three slides of each sample were analyzed for each method, thus totalizing 2,610 slides examined. The frequency of strongloidiasis in the total alcoholic group (33.3%) and in the subgroups of alcoholics, i.e., patients with hepatic cirrhosis (44.4%), with chronic pancreatitis (33.3%), and those with no cirrhosis or pancreatitis (29.6%) was statistically higher than that found in the control group (5.5%). None of the individuals with nonalcoholic hepatic cirrhosis had S. stercoralis infection. Our results showed that the chronic alcoholism itself is an important factor that predisposes to strongyloidiasis.     

DR antigens and rheumatoid arthritis: a study of two populations.

Thr prevalence of HLA-DR antigens was determined in a group of white patients with rheumatoid arthritis, similar patients originating from the Indian subcontinent, and corresponding controls. Rheumatoid arthritis was found to be highly associated with DR4 in the white patients but with DR1 in the Indian patients. These results raise the possibility that the DR antigens themselves do not play a part in increasing susceptibility to rheumatoid arthritis, but the locus for increased susceptibility is probably closely linked to the DR locus.     

Insulin resistance in Fibrocalculous (tropical) pancreatic diabetes

Insulin resistance was assessed by Insulin Tolerance Test (ITT) in 12 patients with FCPD, 10 with NIDDM and 12 age and sex matched control subjects. The mean BMI of the FCPD was lower than the NIDDM and control groups (P less than 0.001). There was no significant difference between the mean fasting plasma glucose or HbA1 between the FCPD and NIDDM patients. The mean fasting C-peptide of the FCPD group was significantly lower than the NIDDM and control groups (P less than 0.001). The mean glucose disposal rate (KITT) was 5.57 +/- 2.28 in the control group, 2.15 +/- 2.00 in the FCPD and 1.77 +/- 0.91 in the NiDDM group (P less than 0.001, control vs FCPD and NIDDM). The difference in KITT between FCPD and NIDDM groups was not significant statistically. The data suggests that patients with FCPD have evidence of insulin resistance and this is similar to that seen in NIDDM patients.     

Predictors of Osteodystrophy in Patients with Chronic Nonalcoholic Pancreatitis with or without Diabetes

ObjectiveTo study bone mineral content (BMC), bone mineral density (BMD), vitamin D status, and bone mineral variables in patients with chronic nonalcoholic pancreatitis and to determine the relationship between pancreatic dysfunction and these variables.MethodsThirty-one eligible nonalcoholic men with proven chronic pancreatitis and 35 male control subjects were studied. Biochemical data, variables of malabsorption, and BMD of the lumbar spine were evaluated.ResultsIn patients with chronic pancreatitis, the mean body mass index (BMI) was 18.46 kg/m² and the median 25-hydroxyvitamin D value was 15.5 (range, 5.0 to 52.0) ng/mL. A T-score of less than -2.5 was found in a higher proportion of study patients (9 of 31, 29%) than of control subjects (3 of 35, 9%). BMI correlated significantly with BMC (r = 0.426; P = .017). There was an inverse correlation between stool fat and BMC (r = -0.47; P = .03) in patients with chronic pancreatitis and steatorrhea. There was no significant correlation between serum 25-hydroxyvitamin D or biochemical variables and BMD. Patients with steatorrhea had a significantly lower BMC than did those without steatorrhea, and this difference could not be accounted for by differences in BMI, presence of diabetes, or hypovitaminosis D.ConclusionPancreatic osteodystrophy is a novel entity consisting of osteopenia, osteoporosis, and osteomalacia in patients with chronic pancreatitis. The inverse correlation between stool fat and BMC in patients with chronic pancreatitis, the strong positive correlation between BMI and BMC, and the lack of difference in BMC between subjects with vitamin D sufficiency and those with vitamin D deficiency suggest that long-standing malabsorption with attendant chronic undernutrition is the major factor contributing to the changes in BMC. (Endocr Pract. 2011;17:897-905)     

Effect of Bromocriptine-Qr On Glycemic Control in Subjects with Uncontrolled Hyperglycemia On One or Two Oral Anti-Diabetes Agents

ObjectiveTo investigate the effect of Bromocriptine QR on glycemic control in patients with type 2 diabetes whose glycemia is poorly controlled on one or two oral anti-diabetes agents.MethodsFive hundred fifteen Type 2 Diabetes Mellitus (T2DM) subjects (ages 18 to 80 and average body mass index [BMI] of 32.7) with baseline HbA1c ≥ 7.5 and on one or two oral anti-diabetes (OAD) medications (metformin, sulfonylurea, and/or thiazolidinediones) were randomized 2:1 to bromocriptine-QR (1.6 to 4.8 mg/day) or placebo for a 24 week treatment period. Study investigators were allowed to adjust, if necessary, subject anti diabetes medications during the study to attempt to achieve glycemic control in case of glycemic deterioration. The impact of bromocriptine-QR treatment intervention on glycemic control was assessed in subjects on any one or two OADs (ALL treatment category) (N = 515), or on metformin with or without another OAD (Met/OAD treatment category) (N = 356), or on metformin plus a sulfonylurea (Met/SU treatment category) (N = 245) 1) by examining the between group difference in change from baseline a) concomitant OAD medication changes during the study, and b) HbA1c and 2) by determining the odds of reaching HbA1c of ≤ 7.0% on bromocriptine-QR versus placebo.ResultsSignificantly more patients (approximately 1.5 to 2-fold more; P < .05) intensified concomitant anti diabetes medication therapy during the study in the placebo versus the bromocriptine-QR arm. In subjects that did not change the intensity of the baseline diabetes therapy (72%), and that were on any one or two OADs (ALL), or on metformin with or without another OAD (Met/OAD), or on metformin plus sulfonylurea (Met/SU), the HbA1c change for bromocriptine-QR versus placebo was − 0.47 versus + 0.22 (between group delta of − 0.69, P < .0001), − 0.55 versus + 0.26 (between group delta of − 0.81, P < .0001) and − 0.63 versus + 0.20 (between group delta of − 0.83, P < .0001) respectively, after 24 weeks on therapy. The odds ratio of reaching HbA1c of ≤ 7.0% was 6.50, 12.03 and 11.45 (P < .0002) for these three groups, respectively.ConclusionIn T2DM subjects whose hyperglycemia is poorly controlled on one or two oral agents, bromocrip tine-QR therapy for 24 weeks can provide significant added improvement in glycemic control relative to adding placebo. (Endocr Pract. 2012;18:931-943)     

Insulin sensitivity in pancreatitis, liver diseases, steroid treatment and hyperthyroidism assessed by glucose, insulin and somatostatin infusion

In order to assess insulin sensitivity for glucose utilization in the other type of diabetes, insulin sensitivity tests were performed in subjects with pancreatitis, liver disease, steroid treatment and hyperthyroidism. Insulin sensitivity for glucose utilization decreased in subjects with liver disease, steroid treatment and hyperthyroidism irrespective of the presence or absence of glucose intolerance. Hyperinsulinism was associated in most of the subjects with liver disease and steroid treatment, but even in normo-insulinemic subjects, insulin insensitivity was observed. Obesity was associated with only 2 cases in both pancreatitis and liver diseases and therefore was excluded as a major cause for insulin insensitivity in subjects studied. In subjects with pancreatitis, insulin sensitivity was not significantly decreased. It is to be noted that 4 out of 5 subjects with diabetic OGTT (oral glucose tolerance test) exhibited normal insulin sensitivity. The results indicate that in pancreatitis, tissue insulin sensitivity for glucose metabolism is not altered and therefore can be used as a marker to differentiate the other type of diabetes due to pancreatitis from type 1 or 2 diabetes. Although hyperinsulinemia may be attributable to insulin insensitivity in subjects studied at least in part, steroid and thyroid hormone are thought to act directly antagonistically with insulin for glucose metabolism.     

Association of Obesity,but not Diabetes or Hypertension,with Glucocorticoid Receptor N363S Variant

Objective: To determine whether the N363S variant in the glucocorticoid receptor (encoded by nuclear receptor subfamily 3, group C, member 1: NR3C1) is associated with obesity, type 2 diabetes, or hypertension. Research Methods and Procedures: This was a cross‐sectional case‐control study involving 951 Anglo‐Celtic/Northern European subjects from Sydney. This study consisted of the following: 1) an obesity clinic group, most of whom had “morbid obesity” (mean BMI for group = 43 ± 8 kg/m²; n = 152); 2) a type 2 diabetes clinic group (n = 356); 3) patients with essential hypertension who had a strong family history (n = 141); and 4) normal healthy controls (n = 302). N363S genotype, BMI, and a range of other parameters relevant to each group were measured. Results: Compared with the frequency of 0.04 in nonobese healthy subjects, the S363 allele was significantly higher in obesity clinic patients (0.17; p = 5.6 × 10^?8), subjects with diabetes who were also obese (0.09; p = 0.0045), subjects with hypertension who were also overweight (0.08; p = 0.0016), and overweight healthy subjects (0.12; p = 0.0004). Discussion: The NR3C1 N363S variant is associated with obesity and overweight in a range of patient settings but is not associated with hypertension or type 2 diabetes.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.