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PNPLA3 Gene Polymorphisms Associated with NAFLD Patients in India
NON-ALCOHOLIC FATTY LIVER DISEASE
NAFLD
Background: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease that occurs due to accumulation of fat in liver. The disease is commonly associated with obesity, but prevalence of NAFLD is increasing in non-obese Indian population nowadays. It is postulated that insulin resistance, small bowel bacterial overgrowth, increased intestinal permeability and low-grade endotoxemia may be responsible for steatosis and pathogenesis of NAFLD. The present study was undertaken to evaluate SIBO, endotoxemia and insulin resistance in lean and obese NAFLD patients. Methodology: The prevalence of SIBO was determined using the glucose hydrogen breath test (GHBT). The SIBO was diagnosed positive if the fasting breath hydrogen (H2) concentration was >20 ppm or there was increase of H2 levels >12 ppm over the baseline following ingestion of glucose. The lipopolysaccharide (LPS)-binding protein (LBP) was determined by ELISA. HOMA-IR that measures insulin resistance was assessed by taking fasting serum insulin and fasting blood glucose into consideration. Results: The prevalence of SIBO was determined by GHBT done in171 consecutive NAFLD patients and percentage prevalence was 20.5%. The NAFLD patients were further stratified to groups BMI <25 and BMI >25 and percentage prevalence was 4% (<25 BMI, n = 25), and 18% (>25 BMI, n = 29), respectively and 0% in disease control (CHB, n = 25) and healthy control (n = 16) group, respectively. The LBP is synthesized by hepatocytes and intestinal epithelial cells. Normal LBP serum concentrations are 5–10 mg/ml and may rise up to 200 mg/ml immediately during induction of an acute-phase response. We have observed LBP level within normal range and among the groups, the mean concentrations in the serum are as follows: 7.91 0.73 mg/ml (<25 BMI, n = 8), 6.62 1.56 mg/ml (>25 BMI, n = 8), 4.0 1.88 mg/ml (CHB, n = 6), 2.96 1.24 mg/ml (healthy, n = 5). As per IDF guidelines for metabolic syndrome, increased risks of insulin resistance (HOMA IR >1.6–2.5) are 32% (<25 BMI, n = 25), 34% (>25 BMI, n = 29), 13% (CHB, n = 25) and 0% (Healthy, n = 16). The insulin resistant (HOMA IR >2.5) prevalence among groups are 24% (<25 BMI, n = 25), 52% (>25 BMI, n = 29), 9% (CHB, n = 25) and 0% (Healthy, n = 16). Conclusion: We have observed higher prevalence of SIBO in >25 BMI NAFLD group than that of the <25 BMI group. There is also prevalence (3.5%) of methanogenic bacteria in NAFLD population. The LBP may be considered as surrogate marker for SIBO in GHBT negative cases. Increased risk of insulin resistance was also observed in lean NAFLD patients when compared to obese NAFLD patients. S24
Corresponding author: Baibaswata Nayak. E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.265
COMPARISON OF ALT LEVELS AND BMI IN PATIENTS WITH CHRONIC HEPATITIS B AND NON ALCOHOLIC STEATOHEPATITIS S. Ramakrishna Lakeshore Hospital and Research Centre, Kochi, India
Introduction: Elevated transaminase is a feature of hepatitis. ALT levels are elevated and guide therapy in both chronic hepatitis B (CHB) and Non-alcoholic Steatohepatitis (NASH). Aim: To compare ALT levels and BMI in patients with CHB and NASH. Methods: A retrospective cross-sectional study was done from Jan 2006 to Dec 2014 which included 22 patients (18 M, 4 F) of NASH and 26 patients (17 M, 9 F) of CHB who underwent liver biopsy at Lakeshore hospital and research centre, Kochi. The BMI and ALT levels were taken the day before or after liver biopsy. Those with incomplete data were excluded. Results: The ALT was 147 + 91 (mean + SD) and 98 69 respectively in NASH and CHB (P-value = 0.02). The BMI 27 4 & 26 4 and age 41 9 & 41 10 were similar in both NASH and CHB. M:F ratio 4.5:1.0 in NASH compared to 1.8:1.0 in CHB was not significant. 14/22 (63%) NASH and 12/26 (46%) of CHB were overweight/obese. 20 NASH and 23 CHB had one or more features of the metabolic syndrome. Conclusions: Although the ALT was significantly higher in NASH, in many patients, it is not possible to differentiate CHB from NASH by common biochemical tests. http://dx.doi.org/10.1016/j.jceh.2015.07.266
PNPLA3 GENE POLYMORPHISMS ASSOCIATED WITH NAFLD PATIENTS IN INDIA Manvi Vernekar 1,2, Deepak N. Amarapurkar 1,2, 1
Bombay Hospital, Mumbai, Mumbai, India, Technology, Mumbai, India
2
Institute of Chemical
Background: Non Alcoholic Fatty Liver Disease (NAFLD) is considered to be the hepatic © 2015, INASL
JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
Table 1 Gene
SNP
Allele
Cases (n)
Controls 2 OR 95% (n) CI
PNPLA3 Rs738409 C 36 G 10
69
6
0.03
3.19
1.07–9.50
Conclusion: Corroborating with the data, the G allele of rs738409 was found to be associated with NAFLD in Indian subjects. Corresponding author: . E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.267
SMALL INTESTINAL BACTERIAL OVERGROWTH AND TOLL LIKE RECEPTOR SIGNALING IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE Shweta Kapil, Ajay Duseja, Bal Sharma, Bhupesh Singla, Anuradha Chakraborti, Ashim Das, Pallab Ray, Radha K. Dhiman, Yogesh Chawla Postgraduate Institute of Medical Education and Research, Chandigarh, India
Introduction: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD. Methodology: 142 subjects comprised of NAFLD (n = 60, mean age 38.7 10.4 yrs), chronic viral hepatitis (CVH) (n = 32, mean age 36.56 4.2 yrs) and healthy volunteers (HVs) (n = 50, mean age 36.56 4.2 yrs). Duodenal fluid was taken endoscopically in 32 prospective patients with NAFLD for evaluation of SIBO. Hepatic mRNA expression of TLR4, CD14, TLR2, NF-kb, and MD2 and protein expression of TLR4 and TLR2 were studied in 64 patients (NAFLD = 32, CVH = 32) by RT-PCR and immunohistochemistry, respectively. Serum levels of TNF-a, adiponectin, insulin and endotoxins were also evaluated. Results: SIBO was present in 12 (37.5%) out of 32 patients with NAFLD with Escherichia coli as the predominant bacterium. In comparison to those without SIBO, patients with SIBO had significantly higher endotoxin levels, higher CD14 mRNA, NF-kb mRNA, and TLR4 protein expression. Patients with NASH had significantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison to patients without NASH. Serum levels of TNF-a, endotoxins, and insulin were significantly higher and of adiponectin were lower in NAFLD in comparison to CVH and HVs. Conclusions: Our study provides the first direct evidence of role of SIBO and endotoxemia and its relation with TLR signaling genes and liver histology in patients with NAFLD. Corresponding author: Ajay Duseja. E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.268
Journal of Clinical and Experimental Hepatology | June/July 2015 | Vol. 5 | No. S2 | S21–S26
S25
NAFLD
manifestation of the Metabolic Syndrome. However, genetic predisposition of NAFLD has not been extensively studied. Single Nucleotide Polymorphisms (SNPs) in Patatin like phospholipase domain containing protein 3 (PNPLA3) gene Rs738409 are known to be associated with NAFLD. Aim: To determine the association of PNPLA3 rs738409 gene polymorphism in Indian NAFLD patients and healthy controls. Methods: Subjects were recruited into control (n = 75 with normal ultrasound evaluation) and case (n = 46 with ultrasound proven fatty infiltration of liver) groups. Biochemical parameters like lipid and liver profiling were carried out. Anthropometric measures like age, weight, height and BMI were calculated. Polymerise Chain Reaction (PCR) using specific primers with subsequent Restriction Fragment Length Polymorphisms (RFLP) technique was employed for genotyping. Results: A total of 121 subjects were recruited with 75 controls (mean age 40.18 12.53 years) and 46 cases (mean age 41.9 10.1 years). BMI of cases (26.49 0.76) was higher than that of controls (22.82 0.53) (P < 0.05). AST levels were also significantly elevated in cases (P < 0.05). Rs738409 was found to be in genotypic (P = 0.05) and allelic (P < 0.05) association with NAFLD. The frequency of minor allele G in cases was significantly higher than in the control groups. The results showed that the G/G genotype was more likely to lead to NAFLD (OR 3.19, 95% CI 1.07–9.50) (Table 1).
NAFLD
Background: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease that occurs due to accumulation of fat in liver. The disease is commonly associated with obesity, but prevalence of NAFLD is increasing in non-obese Indian population nowadays. It is postulated that insulin resistance, small bowel bacterial overgrowth, increased intestinal permeability and low-grade endotoxemia may be responsible for steatosis and pathogenesis of NAFLD. The present study was undertaken to evaluate SIBO, endotoxemia and insulin resistance in lean and obese NAFLD patients. Methodology: The prevalence of SIBO was determined using the glucose hydrogen breath test (GHBT). The SIBO was diagnosed positive if the fasting breath hydrogen (H2) concentration was >20 ppm or there was increase of H2 levels >12 ppm over the baseline following ingestion of glucose. The lipopolysaccharide (LPS)-binding protein (LBP) was determined by ELISA. HOMA-IR that measures insulin resistance was assessed by taking fasting serum insulin and fasting blood glucose into consideration. Results: The prevalence of SIBO was determined by GHBT done in171 consecutive NAFLD patients and percentage prevalence was 20.5%. The NAFLD patients were further stratified to groups BMI <25 and BMI >25 and percentage prevalence was 4% (<25 BMI, n = 25), and 18% (>25 BMI, n = 29), respectively and 0% in disease control (CHB, n = 25) and healthy control (n = 16) group, respectively. The LBP is synthesized by hepatocytes and intestinal epithelial cells. Normal LBP serum concentrations are 5–10 mg/ml and may rise up to 200 mg/ml immediately during induction of an acute-phase response. We have observed LBP level within normal range and among the groups, the mean concentrations in the serum are as follows: 7.91 0.73 mg/ml (<25 BMI, n = 8), 6.62 1.56 mg/ml (>25 BMI, n = 8), 4.0 1.88 mg/ml (CHB, n = 6), 2.96 1.24 mg/ml (healthy, n = 5). As per IDF guidelines for metabolic syndrome, increased risks of insulin resistance (HOMA IR >1.6–2.5) are 32% (<25 BMI, n = 25), 34% (>25 BMI, n = 29), 13% (CHB, n = 25) and 0% (Healthy, n = 16). The insulin resistant (HOMA IR >2.5) prevalence among groups are 24% (<25 BMI, n = 25), 52% (>25 BMI, n = 29), 9% (CHB, n = 25) and 0% (Healthy, n = 16). Conclusion: We have observed higher prevalence of SIBO in >25 BMI NAFLD group than that of the <25 BMI group. There is also prevalence (3.5%) of methanogenic bacteria in NAFLD population. The LBP may be considered as surrogate marker for SIBO in GHBT negative cases. Increased risk of insulin resistance was also observed in lean NAFLD patients when compared to obese NAFLD patients. S24
Corresponding author: Baibaswata Nayak. E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.265
COMPARISON OF ALT LEVELS AND BMI IN PATIENTS WITH CHRONIC HEPATITIS B AND NON ALCOHOLIC STEATOHEPATITIS S. Ramakrishna Lakeshore Hospital and Research Centre, Kochi, India
Introduction: Elevated transaminase is a feature of hepatitis. ALT levels are elevated and guide therapy in both chronic hepatitis B (CHB) and Non-alcoholic Steatohepatitis (NASH). Aim: To compare ALT levels and BMI in patients with CHB and NASH. Methods: A retrospective cross-sectional study was done from Jan 2006 to Dec 2014 which included 22 patients (18 M, 4 F) of NASH and 26 patients (17 M, 9 F) of CHB who underwent liver biopsy at Lakeshore hospital and research centre, Kochi. The BMI and ALT levels were taken the day before or after liver biopsy. Those with incomplete data were excluded. Results: The ALT was 147 + 91 (mean + SD) and 98 69 respectively in NASH and CHB (P-value = 0.02). The BMI 27 4 & 26 4 and age 41 9 & 41 10 were similar in both NASH and CHB. M:F ratio 4.5:1.0 in NASH compared to 1.8:1.0 in CHB was not significant. 14/22 (63%) NASH and 12/26 (46%) of CHB were overweight/obese. 20 NASH and 23 CHB had one or more features of the metabolic syndrome. Conclusions: Although the ALT was significantly higher in NASH, in many patients, it is not possible to differentiate CHB from NASH by common biochemical tests. http://dx.doi.org/10.1016/j.jceh.2015.07.266
PNPLA3 GENE POLYMORPHISMS ASSOCIATED WITH NAFLD PATIENTS IN INDIA Manvi Vernekar 1,2, Deepak N. Amarapurkar 1,2, 1
Bombay Hospital, Mumbai, Mumbai, India, Technology, Mumbai, India
2
Institute of Chemical
Background: Non Alcoholic Fatty Liver Disease (NAFLD) is considered to be the hepatic © 2015, INASL
JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
Table 1 Gene
SNP
Allele
Cases (n)
Controls 2 OR 95% (n) CI
PNPLA3 Rs738409 C 36 G 10
69
6
0.03
3.19
1.07–9.50
Conclusion: Corroborating with the data, the G allele of rs738409 was found to be associated with NAFLD in Indian subjects. Corresponding author: . E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.267
SMALL INTESTINAL BACTERIAL OVERGROWTH AND TOLL LIKE RECEPTOR SIGNALING IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE Shweta Kapil, Ajay Duseja, Bal Sharma, Bhupesh Singla, Anuradha Chakraborti, Ashim Das, Pallab Ray, Radha K. Dhiman, Yogesh Chawla Postgraduate Institute of Medical Education and Research, Chandigarh, India
Introduction: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD. Methodology: 142 subjects comprised of NAFLD (n = 60, mean age 38.7 10.4 yrs), chronic viral hepatitis (CVH) (n = 32, mean age 36.56 4.2 yrs) and healthy volunteers (HVs) (n = 50, mean age 36.56 4.2 yrs). Duodenal fluid was taken endoscopically in 32 prospective patients with NAFLD for evaluation of SIBO. Hepatic mRNA expression of TLR4, CD14, TLR2, NF-kb, and MD2 and protein expression of TLR4 and TLR2 were studied in 64 patients (NAFLD = 32, CVH = 32) by RT-PCR and immunohistochemistry, respectively. Serum levels of TNF-a, adiponectin, insulin and endotoxins were also evaluated. Results: SIBO was present in 12 (37.5%) out of 32 patients with NAFLD with Escherichia coli as the predominant bacterium. In comparison to those without SIBO, patients with SIBO had significantly higher endotoxin levels, higher CD14 mRNA, NF-kb mRNA, and TLR4 protein expression. Patients with NASH had significantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison to patients without NASH. Serum levels of TNF-a, endotoxins, and insulin were significantly higher and of adiponectin were lower in NAFLD in comparison to CVH and HVs. Conclusions: Our study provides the first direct evidence of role of SIBO and endotoxemia and its relation with TLR signaling genes and liver histology in patients with NAFLD. Corresponding author: Ajay Duseja. E-mail: [email protected] http://dx.doi.org/10.1016/j.jceh.2015.07.268
Journal of Clinical and Experimental Hepatology | June/July 2015 | Vol. 5 | No. S2 | S21–S26
S25
NAFLD
manifestation of the Metabolic Syndrome. However, genetic predisposition of NAFLD has not been extensively studied. Single Nucleotide Polymorphisms (SNPs) in Patatin like phospholipase domain containing protein 3 (PNPLA3) gene Rs738409 are known to be associated with NAFLD. Aim: To determine the association of PNPLA3 rs738409 gene polymorphism in Indian NAFLD patients and healthy controls. Methods: Subjects were recruited into control (n = 75 with normal ultrasound evaluation) and case (n = 46 with ultrasound proven fatty infiltration of liver) groups. Biochemical parameters like lipid and liver profiling were carried out. Anthropometric measures like age, weight, height and BMI were calculated. Polymerise Chain Reaction (PCR) using specific primers with subsequent Restriction Fragment Length Polymorphisms (RFLP) technique was employed for genotyping. Results: A total of 121 subjects were recruited with 75 controls (mean age 40.18 12.53 years) and 46 cases (mean age 41.9 10.1 years). BMI of cases (26.49 0.76) was higher than that of controls (22.82 0.53) (P < 0.05). AST levels were also significantly elevated in cases (P < 0.05). Rs738409 was found to be in genotypic (P = 0.05) and allelic (P < 0.05) association with NAFLD. The frequency of minor allele G in cases was significantly higher than in the control groups. The results showed that the G/G genotype was more likely to lead to NAFLD (OR 3.19, 95% CI 1.07–9.50) (Table 1).