Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and Implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.

Development of Food Group Tree-Based Analysis and Its Association with Non-Alcoholic Fatty Liver Disease (NAFLD) and Co-Morbidities in a South Indian Population: A Large Case-Control Study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a global problem growing in parallel to the epidemics of obesity and diabetes, with South Asians being particularly susceptible. Nutrition and behaviour are important modifiers of the disease; however, studies to date have only described dietary patterns and nutrients associated with susceptibility to NAFLD. METHODS: This cross-sectional case-control study included 993 NAFLD patients and 973 healthy controls from Trivandrum (India). Dietary data was collected using a locally validated food frequency questionnaire. A tree-based classification categorised 2165 ingredients into three levels (food groups, sub-types, and cooking methods) and intakes were associated with clinical outcomes. RESULTS: NAFLD patients had significantly higher consumption of refined rice, animal fat, red meat, refined sugar, and fried foods, and had lower consumption of vegetables, pulses, nuts, seeds, and milk compared to controls. The consumption of red meat, animal fat, nuts, and refined rice was positively associated with NAFLD diagnosis and the presence of fibrosis, whereas consumption of leafy vegetables, fruits, and dried pulses was negatively associated. Fried food consumption was positively associated with NAFLD, whilst boiled food consumption had a negative association. Increased consumption of animal fats was associated with diabetes, hypertension, and cardiovascular outcomes among those with NAFLD, whereas consumption of wholegrain rice was negatively associated with these clinical-related outcomes. CONCLUSIONS: The tree-based approach provides the first comprehensive method of classifying food intakes to enable the identification of specific dietary factors associated with NAFLD and related clinical outcomes. This could inform culturally sensitive dietary guidelines to reduce risk of NAFLD development and/or its progression.

High faecal calprotectin levels in intestinal tuberculosis are associated with granulomas in intestinal biopsies.

BACKGROUND: The diagnosis of intestinal tuberculosis (ITB) is sometimes difficult to establish and requires endoscopic investigation with biopsies for histopathological examination. This study aimed to evaluate calprotectin as a marker of inflammation in ITB. METHODS: Patients with ITB were prospectively recruited in Southern India from October 2009 until July 2012. Demographic, clinical, endoscopic and histological features were examined along with faecal calprotectin (FC), serum calprotectin (SC) and C-reactive protein (CRP). RESULTS: Thirty patients (median age 34.5 years, 19 men) were included. Clinical features were abdominal pain (97%), weight loss (83%), cachexia (75%), fatigue (63%), watery diarrhoea (62%), nausea (55%) and fever (53%). Endoscopy showed transverse ulcers (61%), nodularity of mucosa (55%), aphthous ulcers (39%), strictures (10%) and fissures (10%). The terminal ileum and right colon harboured 81% of the lesions. Histology revealed granulomas in biopsies from 10 of the patients. FC and CRP levels showed a strong positive correlation (rs = 0.70, p < 0.01). FC, SC and CRP levels were higher in the granulomatous than the non-granulomatous patients, respectively (median FC 988 µg/g, interquartile range (IQR) 940 vs 87 µg/g, IQR 704, p < 0.01; median SC 8.2 µg/ml, IQR 7.3 vs 3.8 µg/ml, IQR 8.9, p = 0.23; median CRP 38.8 mg/L, IQR 42.9 vs 2.3 mg/L, IQR 13.5, p < 0.01). Higher median calprotectin and CRP levels were detected in patients with extensive than localized disease, but the differences did not reach statistical significance. CONCLUSION: ITB patients with granulomas on histology have high levels of faecal calprotectin and CRP.

Faecal calprotectin levels differentiate intestinal from pulmonary tuberculosis: An observational study from Southern India.

BACKGROUND: Current methods to establish the diagnosis of intestinal tuberculosis are inadequate. OBJECTIVES: We aimed to determine the clinical features of intestinal tuberculosis and evaluate inflammatory biomarkers in intestinal as well as pulmonary tuberculosis. METHODS: We recruited 38 intestinal tuberculosis patients, 119 pulmonary tuberculosis patients and 91 controls with functional gastrointestinal disorders between October 2009 and July 2012 for the investigation of clinical features, C-reactive protein (CRP), faecal and serum calprotectin. Faecal calprotectin ≥200 µg/g was used as a cut-off to determine intestinal inflammation of clinical significance. Three patient categories were established: (a) pulmonary tuberculosis and faecal calprotectin <200 µg/g (isolated pulmonary tuberculosis); (b) pulmonary tuberculosis and faecal calprotectin ≥200 µg/g (combined pulmonary and intestinal tuberculosis); (c) isolated intestinal tuberculosis. RESULTS: Common clinical features of intestinal tuberculosis were abdominal pain, fatigue, weight loss and watery diarrhoea. Intestinal tuberculosis patients had elevated median CRP (10.7 mg/l), faecal calprotectin (320 µg/g) and serum calprotectin (5.7 µg/ml). Complete normalisation of CRP (1.0 mg/L), faecal calprotectin (16 µg/g) and serum calprotectin (1.4 µg/ml)) was seen upon clinical remission. Patients with combined pulmonary and intestinal tuberculosis had the highest levels of CRP (53.8 mg/l) and serum calprotectin (6.5 µg/ml) and presented with signs of more severe disease. CONCLUSION: Calprotectin analysis reveals intestinal tuberculosis in patients with pulmonary tuberculosis and pinpoints those in need of rigorous follow-up.

Routine diagnosis of intestinal tuberculosis and Crohn's disease in Southern India.

AIM: To investigate whether routinely measured clinical variables could aid in differentiating intestinal tuberculosis (ITB) from Crohn's disease (CD). METHODS: ITB and CD patients were prospectively included at four South Indian medical centres from October 2009 to July 2012. Routine investigations included case history, physical examination, blood biochemistry, ileocolonoscopy and histopathological examination of biopsies. Patients were followed-up after 2 and 6 mo of treatment. The diagnosis of ITB or CD was re-evaluated after 2 mo of antituberculous chemotherapy or immune suppressive therapy respectively, based on improvement in signs, symptoms and laboratory variables. This study was considered to be an exploratory analysis. Clinical, endoscopic and histopathological features recorded at the time of inclusion were subject to univariate analyses. Disease variables with sufficient number of recordings and P < 0.05 were entered into logistic regression models, adjusted for known confounders. Finally, we calculated the odds ratios with respective confidence intervals for variables associated with either ITB or CD. RESULTS: This study included 38 ITB and 37 CD patients. Overall, ITB patients had the lowest body mass index (19.6 vs 22.7, P = 0.01) and more commonly reported weight loss (73% vs 38%, P < 0.01), watery diarrhoea (64% vs 33%, P = 0.01) and rural domicile (58% vs 35%, P < 0.05). Endoscopy typically showed mucosal nodularity (17/31 vs 2/37, P < 0.01) and histopathology more frequently showed granulomas (10/30 vs 2/35, P < 0.01). The CD patients more frequently reported malaise (87% vs 64%, P = 0.03), nausea (84% vs 56%, P = 0.01), pain in the right lower abdominal quadrant on examination (90% vs 54%, P < 0.01) and urban domicile (65% vs 42%, P < 0.05). In CD, endoscopy typically showed involvement of multiple intestinal segments (27/37 vs 9/31, P < 0.01). Using logistic regression analysis we found weight loss and nodularity of the mucosa were independently associated with ITB, with adjusted odds ratios of 8.6 (95%CI: 2.1-35.6) and 18.9 (95%CI: 3.5-102.8) respectively. Right lower abdominal quadrant pain on examination and involvement of ≥ 3 intestinal segments were independently associated with CD with adjusted odds ratios of 10.1 (95%CI: 2.0-51.3) and 5.9 (95%CI: 1.7-20.6), respectively. CONCLUSION: Weight loss and mucosal nodularity were associated with ITB. Abdominal pain and excessive intestinal involvement were associated with CD. ITB and CD were equally common.

Metadoxine Versus Placebo for the Treatment of Non-alcoholic Steatohepatitis: A Randomized Controlled Trial.

OBJECTIVE AND DESIGN: The study aimed at assessing the therapeutic efficacy and safety of metadoxine versus placebo on the ultrasonographic and histological features of non-alcoholic steatohepatitis (NASH). SUBJECTS: 134 subjects with biopsy-confirmed NASH were randomized to receive metadoxine 500 mg two times daily (n = 75) or placebo (n = 59) added to the standard of care, over 16 weeks. EFFICACY ENDPOINTS: Originally, the primary efficacy endpoint was the composite of: reduction in the steatosis by ≥1 grade, reduction in hepatic necro-inflammation by ≥1 grade and ALT normalization. Since >50% of patients refused the second biopsy, it was decided to analyze only the individual parameters. RESULTS: There was no significant difference between the treatment and the placebo groups in either liver histology or ALT or AST. Overall, as expected both groups showed reduction in serum ALT and AST compared to baseline. Compared to placebo (9 out 54), patients on metadoxine (34 out of 75) had significantly higher rates of improvement in 1-point in steatosis grade on ultrasound (P-value <0.001). Safety and tolerability did not differ between treatments. CONCLUSION: Metadoxine is not effective in improvement of liver histology or serum ALT or AST in patients with NASH. However, there was significant improvement of steatosis assessed by ultrasound. To properly estimate the effects on histology and transaminases, further studies of longer duration and at higher doses are needed.

Acupressure to reduce labor pain: a randomized controlled trial.

OBJECTIVE: To evaluate the effect of acupressure administered during the active phase of labor on nulliparous women's ratings of labor pain. DESIGN: Randomized controlled trial. SETTING: Public hospital in India. SAMPLE: Seventy-one women randomized to receive acupressure at acupuncture point spleen 6 (SP6) on both legs during contractions over a 30-minute period (acupressure group), 71 women to receive light touch at SP6 on both legs during the same period of time (touch group) and 70 women to receive standard care (standard care group). METHODS: Experience of in-labor pain was assessed by visual analog scale at baseline before treatment, immediately after treatment, and at 30, 60 and 120 minutes after treatment. MAIN OUTCOME MEASURE: Labor pain intensity at different time intervals after treatment compared with before treatment. RESULTS: A reduction of in-labor pain was found in the acupressure group and was most noticeable immediately after treatment (acupressure group vs. standard care group p < 0.001; acupressure group vs. touch group p < 0.001). CONCLUSION: Acupressure seems to reduce pain during the active phase of labor in nulliparous women giving birth in a context in which social support and epidural analgesia are not available. However, the treatment effect is small which suggests that acupressure may be most effective during the initial phase of labor.