Genetic and biochemical characterization of tropical calcific pancreatitis (tcp) and fibrocalculus pancreatic diabetes (fcpd) in Bangladeshi populations

Abstract

Tropical calcific pancreatitis is a form of chronic calcific non alcoholic pancreatitis, seen almost exclusively in developing countries of the tropical world including Bangladesh. Fibrocalculous pancreatic diabetes (FCPD) is a unique form of diabetes secondary to Tropical calcific pancreatitis It has been observed that among all diabetic patients registered in BIRDEM, the central diabetic centre of Diabetic Association of Bangladesh (DAB), about 7% belonged to the group whose onset of diabetes is under 30 years of age. In a cohort of these young (<30 yrs) diabetic patients 13% were found to be constituted by the FCPD patients. To explore the genetic and biochemical characteristics of TCP and FCPD Bangladeshi population the study has been performed at the Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladesh, University of Basel, Switzerland and University of Pittsburg, USA between 2001 and 2008. The study protocol was approved by the local Ethics Committee. Type 2 Diabetes mellitus (T2DM) and Fibrocalculus pancreatic diabetes (FCPD) patients were prospectively selected from the outpatient department of BIRDEM. Tropical calcific pancreatitis (TCP) was chosen from the gastroenterology units at BIRDEM, Bangobandhu Sheik Mujib medical university (BSMMU) in Dhaka and from local gastroenterologists. All the patients were selected within an age band of 30-55 years. 34 TCP, 82 FCPD and 48 Type 2 diabetic subjects have been included in the study. After routine examinations (patient history, physical examination, fasting and 2 hour after breakfast glucose, Glycosolyteted hemoglobin (HbA1c), plain abdominal X-ray) patients with diabetes mellitus and pancreatic calcifications on abdominal X-ray were classified as FCPD and T2DM according to the 1985 WHO classification, which was valid at the time of this investigation. After providing oral informed consent, additional screening investigation was done (anthropometric measurements). The same screening procedures were used for TCP. A modified set of Case Record Form was filled out for each patients and detailed history was taken. Glycemic status has been assessed by fasting glucose and HbA1c. Lipid profile, serum creatinine and serum ALT have been assessed by enzymetric colorimetric method. at BIRDEM. Routine and microscopic examinations of the stool were done to exclude fat, parasites and blood. Arginine stimulation test has been performed at BIRDEM and sample for serum glucose, serum C-peptide and serum glucagon, ICA, IA2, GAD antibody and sample for fecal elastase 1 has been frozen at -200c and later transported to the university of Basel, Switzerland for analysis. The parameters have been analysed by RIA method. Samples for DNA analysis has been also frozen at -20oc and transported to University of Pittsburge, USA. Statistical analysis was performed using Statistical Package for SocialScience (SPSS) for Windows Version 11.0 and p<0.05 was taken as the level of significance throughout. Unpaired‘t’ test. Proportion test, non-parametric tests (eg, Mann-Whitney test, Chi square test and Odds ratio) were applied where applicable. There appeared to be male preponderance in all the groups. Diabetes appears relatively earlier in FCPD than the T2DM subjects which is statistically significant (p<.000). High proportions of TCP and FCPD subjects are from the rural compared to more of the T2DM from urban area (X2=10.842, p= 0.028). Significant difference (p=.945) had been found in WHR value between FCPD and T2DM subjects In TCP subjects, fasting blood glucose (mmol/l, meanSD) and hbA1c (%) is normal as aspected. FCPD subjects show higher fasting blood glucose level than the T2DM which is statistically insignificant. After 2 hours after breakfast blood glucose level in TCP is within normal value, also as expected. FCPD subjects show insignificantly higher blood glucose level than that of T2DM. Age of inset of diabetes (yr) in FCPD is higher than T2DM were showing significant difference statistically (p=.142). Significant difference has been found regarding total cholesterol level in TCP vs FCPD (t/p value=-.004/.997). TCP and FCPD subjects significantly differs in low density lipoprotein (LDL-c) level (mg/dl) ( t/p value=1.588/0.120). Regarding ALT levels, TCP differs significantly with T2DM (p=.019). GAD antibody and IA-2 antibody are absent in all the three groups. ICA anibody has been detected in all the groups. All the groups show severe pancreatic exocrine insufficiency.During Arginine infusion has not altered plasma glucose levels in any of the groups. Arginine stimulation test revealed almost 2 fold increase in serum C-peptide level (nmol/ml) in TCP from base line to 30 min and FCPD subjects showed near 1.5 fold increase of the same compared to that of T2DM which remained un changed. In TCP and FCPD, basal serum level of C-peptide did not differ significantly. The basal value of serum glucagon (pg/ml) revealed the same in TCP, FCPD and T2DM during arginine stimulation test. After 30 minutes of arginine infusion, serum glucagon level showed almost 2 fold increase in both TCP and FCPD with out any significant difference in between confirming preserved  cell function. The association between the SPINK 1N34S gene and TCP and FCPD subjects have been found as expected. The study reports for the first time, the association of CFTR gene in TCP and FCPD subjects. CFTR ex22 gene mutation is not found in any of the TCP subjects. Out of 60 FCPD subjects, 58 (96%) are of wild homozygous CFTR ex22 genotype and 1 (1.7%) heterozygous mutant has been detected and no homozygous mutant have been detected. Among the 33 T2DM subjects, 31 (94%) are of wild homozygous type and no CFTR ex22 mutant have been detected. A number of groups reported the association of CFTR gene mutation in idiopathic chronic pancreatitis but not in TCP and FCPD subjects in particular (Chon et al., 1998; Sharer et al., 1998; Audrezet et al., 2002; Noone et al., 2001; Cohn et al., 2005). The study also reports for the first time, the association of IL-18 gene in TCP, FCPD and T2DM subjects. Although, a significant percentage of IL-18 -607 gene mutation have been found, none of the subjects showed presence of helminth infestation (in regards to the absence of any larva in the stool). This scenario might be due to a wide line of practice of prescribing anthelminthic drug in a regular interval to the poor of rural as well as urban population. The study reports for the first time the correlation of the biochemical parameters and other genetic status of TCP and FCPD subjects with SPINK 1 N34S mutant. Among the biochemical parameters only fecal elastase 1 concentration is significantly lower (p=0.006) compared to the wild genotype. A significant negative correlation is detected between IL-18 -607 CC genotype and the SPINK1 (N34S) haplotype in the subgroup of patients with FCPD ( r=.49 ; p=0.02). 15 TCP and 22 FCPD has either the IL-18 CC genotype and/or the SPINK1 n34S haplotype.