Title: Understanding the molecular mechanisms of hyperinsulinaemic hypoglycaemia and developing novel therapies

Hyperinsulinaemic hypoglycaemia (HH) is a complex medical condition where the beta-cells of the pancreas make too much of the hormone, insulin. This is a very important hormone which controls the blood sugar (glucose) level. Too much insulin in the blood can cause severe low blood glucose levels (hypoglycaemia). Since blood glucose is an essential fuel for the brain, anything that leads to hypoglycaemia will lead to permanent brain damage. HH usually presents in the newborn period with symptomatic (irritability, poor feeding, seizures, coma and sudden death) hypoglycaemia. However there are some types of HH which can present later (in infancy or childhood period). In HH the blood glucose level is often very difficult to control and these patients virtually always require concentrated intravenous dextrose infusions to maintain normal blood glucose levels (normoglycaemia). Great Ormond Street Children's Hospital NHS Trust is a national and international referral center for patients with HH. The Principle Investigator is the clinical lead for the HH service at Great Ormond Street Children's Hospital and the service is commissioned by NHS England.

Certain forms of HH are inherited and have a genetic basis (congenital HH). So far abnormalities in 10 different genetic pathways (genes) have been described which lead to congenital HH. Abnormalities in all these genes account for about 90% of patients who are unresponsive to medical therapy but only about 20% of patients who are medically response. Thus there are a large number of patients with medically responsive forms of congenital HH where the genetic basis is still not known. As part of a MRC funded research project, over the last 3 years we have collected DNA samples (1800) on patients with congenital HH from all over the UK and the world. Each of these patients has been systemically phenotyped and genotyped and all the biochemical data collected. We have identified a unique group pf patients with no known genetic cause of their CHI. In this group of patients we have generated a substantial amount of preliminary data. This patient cohort thus therefore provides us a with unique platform to undertake the proposed research. Understanding the mechanisms of HH in these patients will not only provide novel insights into pancreatic beta-cell physiology but an insight into the more common conditions such as diabetes mellitus.

In some patients with congenital HH the hypoglycaemia is so severe that the only treatment available at the moment is to undertake a major operation to remove nearly the whole pancreas (near total pancreatectomy). However once the pancreas is removed children develop another lifelong and serious condition called diabetes mellitus. Hence there is an urgent need to develop new medical therapies so that we can avoid a near total pancreatectomy. We have recently tried a new oral medication in a very small number of patients (4) with severe Congenital HH who were unresponsive to conventional medical therapies. Interestingly blood sugar levels improved in all these patients in response to this new treatment and they have not undergone a pancreatectomy. These very early observations suggest that this medication might help us in treating other children with CHI. We now want to understand the pharmacology of this medication in children with congenital HH.

In this research study we will use the latest cutting edge techniques in molecular genetics (like exome and genome sequencing) to try and understand the cause of the HH in all those patients where we have not found a genetic cause so far. Using a morphoproteomics approach we will aim to develop new treatment options for children with diffuse and focal fros of congenital HH.

Hyperinsulinaemic hypoglycaemia (HH) is a major cause of recurrent and persistent hypoglycaemia in the neonatal, infancy and childhood periods. So far mutations in ten different genes have been described which lead to HH. The commonest cause is recessive inactivating mutations in ABCC8 and KCNJ11 (which encode the two subunits of the ATP-sensitive potassium (KATP) channels) in beta-cells. In children with medically unresponsive congenital HH mutations in the known genes are found in about 90% of patients. In contrast in children who respond to therapy (diazoxide), mutations in all these genes are only found in about 20% of patients. Thus the aetiology in the vast majority of patients with medically responsive congenital HH is not yet known. We have recruited 1801 patients with HH from 67 countries. 1050 have congenital HH and 1413 were diagnosed in the first 6 months of life. A genetic cause of HH has been found in 43% of patients. We have also described a novel treatment for patients with diffuse HH.

The objectives of this study are to use the state of the art techniques in molecular biology (exome and whole genome sequencing) to try and unravel the molecular basis of congenital HH in those patients where no genetic aetiology has been found and to develop novel medical therapies for children with diffuse and focal HH. We have generated a significant amount of preliminary data on some of these rare HH with complex phenotypes and potentially identified novel causes of HH. We will aim to continue the recruitment and phenotyping of patients with HH. All patients will have genetic testing for known causes of HH (SureSelect capture design now includes all known HH genes (ABCC8, KCNJ11, GCK, HNF4A, HNF1A GLUD1, HADH, SLC16A1, INSR, UCP2, HK1 and CACNA1D)) and if no cause if found then exome/genome sequencing will be undertaken to identify novel causes of HH.

Patients with congenital HH are rare experiments of nature. Studying these patients has already provided unique insights into the mechanism/s that regulate insulin secretion and helped in patient management. For example the identification that recessive inactivating mutations in ABCC8 and KCNJ11 lead to unregulated insulin secretion has shown the pivotal role of KATP channels in regulating insulin secretion. Conversely our the recent discovery that activating mutations in both these genes can lead to neonatal diabetes mellitus (Gloyn AL et al. N Engl J Med. 2004 Apr 29;350(18):1838-49) has completely transformed the lives of many children who are now on oral sulphonylureas (Pearson ER et al. N Engl J Med. 2006 Aug 3;355(5):467-77) rather than daily subcutaneous insulin injections. More recently our discovery that some patients with diffuse congenital HH can be treated with an mTOR inhibitor and thus avoid a pancreatectomy (Senniappan S et al. N Engl J Med. 2014 Mar 20;370(12):1131-7) has also transformed the management of these complex patients. Undertaking a near total pancreatectomy will leave the child with life long diabetes mellitus and pancreatic exocrine insufficiency. Therefore the introduction of drugs which block the mTOR pathway and either reduce or stop insulin production in these patients will bring clinical benefits to children around the world and potentially avoid these children from undergoing a near total pancreatectomy. We have also shown that patients with congenital HH due to mutations in the HADH gene have severe protein induced hypoglycaemia and the hypoglycemia can be relieved by reducing the protein intake in the diet (Heslegrave AJ et al J Clin Endocrinol Metab. 2013 Feb;98(2):496-501). This has again transformed the lives of children with severe HH due to HADH mutations.

Thus studying these patients has the potential to provide further novel insights into the mechanism/s that regulate insulin secretion. Understanding the mechanisms that regulate insulin secretion will have important implications for beta-cell physiology and other common conditions such as diabetes mellitus. Even more importantly revealing these mechanisms will further help in patient management and allow appropriate genetic counseling. The vast majority of patients on diazoxide therapy for congenital HH do not yet have an underlying genetic aetiology. This suggests that novel pathways other than the KATP pathway in the beta-cell are affected in these patients and unraveling these mechanisms will have huge implications for understanding beta-cell physiology. All children on diazoxide therapy for congenital HH develop major side effects such as hirsutisim which is extremely distressing for the child and their family. Thus understanding the molecular mechanism/s of the unregulated insulin secretion in these patients might help us to develop new therapies with less side effects. This again will have a huge impact on the quality of life of these patients.