Understanding the novel genetic mechanisms of congenital hyperinsulinaemic hypoglycaemia.

Background: Hyperinsulinaemic hypoglycaemia (HH) is characterized by unregulated secretion of insulin in the presence of hypoglycaemia. Mutations in nine different genes (ABCC8, KCNJ11, GLUD1, HNF4A, HNF1A, GCK, HADH, SLC16A1 and UCP2) have so far been identified as a cause of HH. Mutations in ABCC8 and KCNJ11, which encode the sulfonylurea receptor 1 (SUR1) and potassium inward-rectifying 6.2 (Kir6.2) subunits of ATP-sensitive potassium channel (KATP channel), are the most common cause of HH. At least two (possibly more) well-described histological subtypes are associated with HH: a focal form and a diffuse form. Diffuse HH are most commonly due to recessive and dominant mutations in ABCC8 and KCNJ11. Focal HH results due to somatic loss of the maternal 11p allele (11p15.1 to 11p15.5) involving the ABCC8 and KCNJ11 region in patients with paternally inherited mutation in ABCC8 or KCNJ11. HH can be transient and resolve within few weeks. Transient HH is seen in association with intrauterine growth restriction, maternal diabetes mellitus, perinatal asphyxia, erythroblastosis fetalis, maternal administration of sulfonylureas, and intravenous glucose infusions during labour. In large series of HH patients, the underlying genetic cause was not identified in approximately 50% of the patients. Whole-exome sequencing is a powerful and cost-effective tool for identifying genetic basis of diseases. It involves sequencing the protein coding regions of the human genome. Aim: To identify novel genetic mechanisms of HH. To functionally characterize two novel KATP channel mutations associated with a unique clinical phenotype. Patients: Four patients had protein-sensitive HH with normal serum ammonia. Two families (one consanguineous and one non-consanguineous) had two affected siblings with HH. All these patients had negative molecular genetics for ABCC8, KCNJ11, GLUD1 and HADH. One patient had a unique phenotype of HH and cardiac arrhythmias. In addition, three patients had two novel KATP channel mutations associated with a unique clinical phenotype (transient HH and combined focal/diffuse HH). Methods: Whole-exome sequencing (WES) was performed on nine patients (from seven families), their parents and unaffected siblings to identify novel, deleterious gene variants. Based on the available biological information, two novel gene variants (p.F108del K2P17 and p.A422V Kv6.2) were considered potential disease causing. The mutations were created in plasmid constructs containing the WT cDNA sequence for KCNK17 (K2P17) and KCNG2 (Kv6.2) using site-directed mutagenesis. These constructs were transfected into HEK293 cells, which were studied by whole-cell patch clamping. Two novel KATP channel mutations (p.T1516M SUR1 and p.*391Rext*94 Kir6.2) were also studied. For studying the p.*391Rext*94 Kir6.2 mutation, the WT human cDNA and 3′UTR sequence of KCNJ11 was cloned into pcDNA 3.1 vector. The KATP channel mutations were created in plasmid construct containing the WT cDNA sequence for ABCC8/KCNJ11 using site-directed mutagenesis. These constructs were transfected into HEK293 cells, which were then studied by whole-cell patch clamping. The p.*391Rext*94 Kir6.2 is a non-stop mutation and the transcripts can undergo degradation by non-stop decay phenomenon. The presence of p. *391Rext*94 Kir6.2 mutant transcript in the pancreatic tissue of the affected patient was studied by cDNA sequencing and Western blotting on the patient’s pancreatic tissue extracted RNA and protein fraction respectively. Results: WES identified two potential disease-causing heterozygous gene variants (p.F108del K2P17 and p.A422V Kv6.2) in a family with a phenotype of HH and cardiac arrhythmia, which were confirmed by Sanger sequencing. Whole-cell patch clamping experiments on HEK293 cells proved both variants to be pathogenic under heterozygous expression. Whole-cell patch clamping experiments on the two novel KATP channel mutations (p.T1516M SUR1 and p.*391Rext*94 Kir6.2) was indicative of pathogenic nature of the mutation. Analysis of the pancreatic tissue, obtained at surgery from the patient with non-stop KCNJ11 mutation (p.*391Rext*94 Kir6.2), by cDNA sequencing and Western blotting established the presence of transcript and protein with non-stop mutation. Conclusions: Mutations in KCNK17 (K2P17) and KCNG2 (Kv6.2) are potential novel genetic mechanisms for HH and cardiac arrhythmias. More patients with similar phenotype need to be screened for mutations in these two genes. A novel mechanism for HH (combined focal and diffuse HH phenotype) and molecular basis for a transient type of HH was identified

Pancreatic endocrine and exocrine function in children following near-total pancreatectomy for diffuse congenital hyperinsulinism.

Published onlineJournal ArticleCONTEXT: Congenital hyperinsulinism (CHI), the commonest cause of persistent hypoglycaemia, has two main histological subtypes: diffuse and focal. Diffuse CHI, if medically unresponsive, is managed with near-total pancreatectomy. Post-pancreatectomy, in addition to persistent hypoglycaemia, there is a very high risk of diabetes mellitus and pancreatic exocrine insufficiency. SETTING: International referral centre for the management of CHI. PATIENTS: Medically unresponsive diffuse CHI patients managed with near-total pancreatectomy between 1994 and 2012. INTERVENTION: Near-total pancreatectomy. MAIN OUTCOME MEASURES: Persistent hypoglycaemia post near-total pancreatectomy, insulin-dependent diabetes mellitus, clinical and biochemical (faecal elastase 1) pancreatic exocrine insufficiency. RESULTS: Of more than 300 patients with CHI managed during this time period, 45 children had medically unresponsive diffuse disease and were managed with near-total pancreatectomy. After near-total pancreatectomy, 60% of children had persistent hypoglycaemia requiring medical interventions. The incidence of insulin dependent diabetes mellitus was 96% at 11 years after surgery. Thirty-two patients (72%) had biochemical evidence of severe pancreatic exocrine insufficiency (Faecal elastase 1<100 µg/g). Clinical exocrine insufficiency was observed in 22 (49%) patients. No statistically significant difference in weight and height standard deviation score (SDS) was found between untreated subclinical pancreatic exocrine insufficiency patients and treated clinical pancreatic exocrine insufficiency patients. CONCLUSIONS: The outcome of diffuse CHI patients after near-total pancreatectomy is very unsatisfactory. The incidence of persistent hypoglycaemia and insulin-dependent diabetes mellitus is very high. The presence of clinical rather than biochemical pancreatic exocrine insufficiency should inform decisions about pancreatic enzyme supplementation

Clinical characteristics and molecular genetic analysis of 22 patients with neonatal diabetes from the South-Eastern region of Turkey: predominance of non-KATP channel mutations.

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThis is an open access article available at http://www.eje-online.org/content/172/6/697.BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare form of monogenic diabetes and usually presents in the first 6 months of life. We aimed to describe the clinical characteristics and molecular genetics of a large Turkish cohort of NDM patients from a single centre and estimate an annual incidence rate of NDM in South-Eastern Anatolian region of Turkey. DESIGN AND METHODS: NDM patients presenting to Diyarbakir Children State Hospital between 2010 and 2013, and patients under follow-up with presumed type 1 diabetes mellitus, with onset before 6 months of age were recruited. Molecular genetic analysis was performed. RESULTS: Twenty-two patients (59% males) were diagnosed with NDM (TNDM-5; PNDM-17). Molecular genetic analysis identified a mutation in 20 (95%) patients who had undergone a mutation analysis. In transient neonatal diabetes (TNDM) patients, the genetic cause included chromosome 6q24 abnormalities (n=3), ABCC8 (n=1) and homozygous INS (n=1). In permanent neonatal diabetes (PNDM) patients, homozygous GCK (n=6), EIF2AK3 (n=3), PTF1A (n=3), and INS (n=1) and heterozygous KCNJ11 (n=2) mutations were identified. Pancreatic exocrine dysfunction was observed in patients with mutations in the distal PTF1A enhancer. Both patients with a KCNJ11 mutation responded to oral sulphonylurea. A variable phenotype was associated with the homozygous c.-331C>A INS mutation, which was identified in both a PNDM and TNDM patient. The annual incidence of PNDM in South-East Anatolian region of Turkey was one in 48 000 live births. CONCLUSIONS: Homozygous mutations in GCK, EIF2AK3 and the distal enhancer region of PTF1A were the commonest causes of NDM in our cohort. The high rate of detection of a mutation likely reflects the contribution of new genetic techniques (targeted next-generation sequencing) and increased consanguinity within our cohort.The genetic testing was funded by the Wellcome Trust (Senior Investigator Award to Profs S Ellard and A T Hattersley), and by Diabetes UK (Project funding to Dr D J Mackay). H Demirbilek was funded by European Society for Paediatric Endocrinology (ESPE) and The Scientific and Technological Research Council of Turkey (TUBITAK) for his 1 year clinical fellowship at University College London (UCL), Institute of Child Health, Great Ormond Street Hospital for Children, NHS Trust, Department of Paediatric Endocrinology

Precipitation-aerosol relationship over the Indian region during drought and excess summer monsoon years

This study investigates the aerosols-rainfall interaction during Indian summer monsoon and characterizes their difference in drought and excess summer monsoon years, based on MODIS (MODerate Resolution Imaging Spectro-radiometer) derived Aerosol Optical Depth (AOD) at 550 nm. AOD has been estimated using Level-2 MODIS Terra Data Version 6. AOD in drought years is found to be higher over India compared to excess monsoon years. The total effect of aerosols causes reduction of summer rainfall but with distinct differences in their impact during strong and weak summer monsoon years, due to the changes in clouds, radiation, large-scale circulation, and convection. Aerosol and cloud characteristics exhibit strong association to rainfall variability in interannual time scales. Variability in cloud effective radius and cloud optical thickness is found to be consistent with aerosol effect

Intranasal inoculation of Chlamydia trachomatis mouse pneumonitis agent induces significant neutrophil infiltration which is not efficient in controlling the infection in mice

Previous studies have shown that chlamydial infection is accompanied by significant infiltration of neutrophils at the site of infection. However, the role of neutrophils in host defence against chlamydial infection is not clearly understood. Using genetically different inbred mouse strains and CXCR-2 gene knockout (KO) mice, we examined the mechanism for neutrophil recruitment and the role of neutrophils during chlamydial lung infection. Our data showed that C3H mice exhibited significantly higher and more persistent neutrophil infiltration in the lung than did C57BL/6 mice following Chlamydia trachomatis mouse pneumonitis infection. The massive neutrophil infiltration in C3H mice was paralleled by high-level expression of CXCR-2 and its ligands, CXC chemokines (macrophage inflammatory protein 2, cytokine-induced neutrophil attractant (KC) and lipopolysaccharide-induced CXC chemokine), and proinflammatory cytokines (tumour necrosis factor-α, interleukin-1 and interleukin-6) in the lung. Although much greater infiltration of neutrophils was observed in C3H mice than in C57BL/6 mice, the former mice had more severe disease and higher in vivo chlamydial growth than the latter. Moreover, CXCR-2 KO mice, which revealed a dramatic reduction in neutrophil activity, showed comparable chlamydial infection to wild-type mice. These results suggest that neutrophils are not efficient for controlling chlamydial lung infection