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Seminars in Neonatology (2004) 9, 49–58 Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny REVIEW ARTICLE Neonatal hypoglycaemia: aetiologies Pascale de Lonlay *, Irina Giurgea, Guy Touati, Jean-Marie Saudubray Department of Paediatrics, Hôpital Necker — Enfants Malades, 149 rue de Sèvres, 75743 Paris cedex 15, France KEYWORDS Hypoglycaemia; Hyperinsulinism; Gluconeogenesis; Fatty acid disorder; Growth hormone; Glucagon; Cortisol; Insulin-like growth factor 1; Glucose transporter disorders Summary Diagnosis of glucose status requires knowledge of the homeostatic mechanisms that maintain the blood glucose concentration between the narrow range of 2.5 and 7.5 mmol/l during periods of eating or fasting. Hypoglycaemia occurring within the first few hours after eating is suggestive of hyperinsulinism. Most glucose is subsequently converted into glycogen in the liver, and hypoglycaemia occurring during this phase is suggestive of glycogenosis. During fasting, gluconeogenesis progressively replaces glycogen as the major source of blood glucose, and hypoglycaemia occurring during this period is suggestive of impaired gluconeogenesis or fatty acid disorders. Growth hormone, glucagon, cortisol and insulin-like growth factor 1 deficiencies may also play a role. Other causes of hypoglycaemia have also been identified recently, namely glucose transporter disorders, respiratory chain disorders and congenital disorders of glycosylation. © 2003 Elsevier Ltd. All rights reserved. Classification of congenital hypoglycaemias Glucose is of essential, fundamental importance for brain metabolism.1 The major source of glucose to the brain is the blood supply, so if the blood glucose content becomes deficient, this may lead to a severe encephalopathy. A prompt diagnosis is essential for the management of hypoglycaemia. This requires knowledge of the homeostatic mechanisms that maintain blood glucose concentration between the relatively narrow range of 2.5 and 7.5 mmol/l during periods of eating or fasting.2 Within the last decade, many new insights and facts have increased our understanding of causes and mechanisms of genetic hypoglycaemia.3 During feeding, the liver builds up energy stores in the form of glycogen and triglyceride, the latter * Correspondence to: P. de Lonlay, Department of Paediatrics, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75743 Paris cedex 15, France. Tel.: +33-144494852; fax: +33-144494850 E-mail address: [email protected] being exported to adipose tissue. During fasting, it releases glucose and ketone bodies. The maintenance of a normal blood glucose level is dependent upon: (1) functionally intact hepatic glycogenolytic and gluconeogenic enzyme systems; (2) an adequate supply of endogenous gluconeogenic substrates (amino acids, glycerol and lactate); (3) an adequate energy supply provided by B-oxidation of fatty acids to synthesize glucose and ketone bodies, the latter being exported to peripheral tissues and used preferentially to glucose as an alternative fuel; and (4) a normal endocrine system for integrating and modulating these processes. The major signals controlling the transition between fed and fasted states are glucose, insulin and glucagon. These directly or indirectly influence the enzymes that regulate liver carbohydrate and fatty acid metabolism, and thereby orient metabolic fluxes towards either energy storage or substrate release.3,4 Based on the origin of glucose in blood, it is possible to divide the timing of glucose homeostasis into five phases. In the first phase (absorptive and 1084-2756/04/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2003.08.002 50 postphase), blood glucose is derived principally from exogenous carbohydrate (glucose ingestion). The concentrations of insulin and glucose are elevated, and glucagon is depressed. Glucose in excess of the fuel needs is stored as glycogen in liver and muscle, or is converted to lipid. This is the only phase in which the liver is a net user of glucose, and gluconeogenesis is of little consequence for glucose homeostasis. Hypoglycaemia occurring during this phase is suggestive of hyperinsulinism. In the second phase, for 3–4 h after glucose ingestion (postabsorptive phase and early starvation), insulin returns to basal levels, glucagon increases and the liver produces glucose, derived principally from stored glycogen. The major user of glucose during this phase is the brain, which oxidizes glucose exclusively. Other obligate glycolysers, such as red blood cells and the renal medulla, also use glucose during this period. Muscles and adipose tissue, however, use glucose at a slower rate than phase. The glycogen present in the liver after an overnight fast (90 g in adults, 20–25 g in a 10-kg infant) is only sufficient to meet the requirements of peripheral tissues for half a day. Hypoglycaemia occurring during this phase is suggestive of glycogenosis. Phase 3 (early/intermediate starvation) begins after 12–16 h of fast. In this third phase, gluconeogenesis progressively replaces glycogen as the major source of blood glucose. It is during this phase, when glycogen stores are depleted and the brain has not yet begun to utilize ketone bodies in significant quantities, that the demand for gluconeogenesis is at its peak, and susceptibility to hypoglycaemia because of impaired gluconeogenesis is greatest. It must be stressed that this period starts immediately after the overnight physiological fast. Hypoglycaemia occurring during this fasting period is suggestive of impaired gluconeogenesis. Fatty acids are essential for gluconeogenesis, which requires energy. In phase 4 (long fasting period), fatty acid disorders are described in which hypoglycaemia is associated with multivisceral failure by acute ATP deficiency. Growth hormone (GH), glucagon, cortisol and insulin-like growth factor 1 (IGF1) deficiencies also play a role and summarize phase 5 in which the endocrine system is effective, whatever the eating and fasting periods. Causes of hypoglycaemia Table 1 lists the causes of genetic hypoglycaemia. In our experience, hyperinsulinism and fatty acid oxidation (FAO) disorders are the most frequent causes of genetic hypoglycaemia. Conversely, P. de Lonlay et al. patients with glycogenosis and endocrine disorders (mostly GH deficiency and adrenal disorders) are not specifically referred to our ward and thus are probably under-represented. Among the rare disorders, ketogenesis and ketolytic defects, glycogen synthetase deficiency, and mitochondrial respiratory chain defects represent interesting and original mechanisms of hypoglycaemia. Hyperinsulinism is a heterogeneous disorder of primary origin, or occurs secondary to fatty acid deficiency, congenital disorders of glycosylation or glutamate dehydrogenase deficiency. The principal differential diagnosis is facticious hypoglycaemia secondary to Munchausen by proxy syndrome, which mimics unusual genetic hypoglycaemia. The following paragraphs will focus on the most frequent aspects of congenital hyperinsulinism, FAO disorders and some other rare enzymatic and glucose transporter disorders (mostly non-ketotic hypoglycaemias). Hyperinsulinism The diagnostic criteria for congenital hyperinsulinism (CI) include fasting and postprandial hypoglycaemia (<2 mmol/l) with hyperinsulianemia (plasma insulin concentrations >3 mU/l), requiring high rates of intravenous glucose infusion (>10 mg/ kg/min) to maintain blood glucose >3 mmol/l, a positive response to the subcutaneous or intramuscular administration of glucagon (plasma glucose concentration increases by 2–3 mmol/l following 0.5 mg glucagon) and persistent hypoglycaemia throughout the first month of life.5,6 In the absence of clearly abnormal insulin levels during hypoglycaemia, a 4–6 h fasting test searching for inappropriately low plasma levels of ketone bodies, free fatty acids and branched chain amino acids may be helpful. However, during the neonatal period, diagnosis is usually clear, mostly based on the severity of hypoglycaemia occurring within 72 h of birth and glucagon responsiveness. The majority of newborns are macrosomic at birth. Hypoglycaemia is always severe, revealed by seizures in about half of all cases, with further brain damage. The rates of intravenous glucose required to prevent hypoglycaemia are elevated, with a mean at 17 mg/kg min in our series. Blood glucose concentrations increase by 2–3 mmol/l in response to subcutaneous or intramuscular administration of glucagon (0.5 mg). A mild hepatomegaly is common and does not exclude the diagnosis of hyperinsulinism. The clinical presentation (hypoglycaemia) is similar whatever the histological lesions and the genetic causes. Most often, no other symptoms are associated with hypoglycaemia. Facial dysmorphism with high forehead, large and bulbous nose Neonatal hypoglycaemia: aetiologies Table 1 51 Genetic hypoglycaemias Diagnosis Postprandial period Hyperinsulinism SUR1 Kir6.2 GLUD1 GK SCHAD CDG Usher Ic (contiguous gene syndrome) BWS Perlman's syndrome Sotos' syndrome A few hours after meal Glycogenosis (I and III) Glycogen synthetase Respiratory chain disorders Fasting period Fructose biphosphatase (neoglucogenesis defect) FAO disorders Ketogenesis and ketolytic defects Functional hypoglycaemia Respiratory chain disorders Fanconi Bickel Endocrinological causes Growth hormone deficiency (GH, IGF1) Adrenal steroid disorders Other GLUT1 (only in CSF) Molecular study Molecular study Enzymatic and molecular study Enzymatic and molecular study Enzymatic and molecular study Western blot of glycosylated protein Molecular study No diagnosis Molecular study Enzymatic and molecular study Enzymatic and molecular study Enzymatic study Enzymatic and molecular study Enzymatic and molecular study Enzymatic and molecular study Enzymatic study Molecular study CSF, cerebrospinal fluid; FAO, fatty acid oxidation; SCHAD, short-chain l-3-hydroxyacyl-CoA dehydrogenase; CDG, congenital disorders of glycosylation; GLUT, glucose transporter; IGF1, insulin-like growth factor 1; GH, growth hormone. with short columella, smooth philtrum and thin upper lip are frequently observed in all types of hyperinsulinism. However, a few cases of syndromic hyperinsulinism have also been described, such as hyperinsulinism associated with Usher's syndrome type Ic or congenital disorders of glycosylation type Ia or Ib. A few patients with Beckwith– Widemann's syndrome, Perlman's syndrome and Sotos' syndrome have also been described. Hyperinsulinaemic hypoglycaemia is due to insulin hypersecretion by the islets of Langerhans. Insulin is the only hormone to lower plasma glucose concentration. It does so by inhibiting glucose release from hepatic glycogen, and increasing glucose uptake in muscle cells. This explains the two main characteristic findings of neonatal CI: the high glucose requirement to correct hypoglycaemia and the responsiveness of hypoglycaemia to exogenous glucagon. Several pathways are involved in the regulation of insulin secretion by the pancreatic B-cells, and this helps to explain the effectiveness of different medical treatments, such as diazoxide, somatostatin, calcium-channel inhibitors and protein-restricted diet (Fig. 1).7,8 Glucose and amino acids stimulate insulin secretion through their metabolism. Glucokinase, the enzyme that initiates glucose metabolism in B-cells, has a high Km for glucose. Thus, the circulating concentrations of glucose directly determine the rate of glucose oxidation. High blood glucose levels increase glucose oxidation and, subsequently, the ATP/ADP ratio, which activates a plasma membrane protein, the sulphonylurea receptor (SUR), and closes an ATP-dependent potassium channel (KATP channel). This leads to depolarization of the B-cell membrane, an influx of extracellular calcium, and the release of insulin from storage granules. Leucine, one of the most potent amino acids to stimulate insulin secretion, acts indirectly as a positive allosteric affector of glutamate dehydrogenase to increase the rate of oxidation of glutamate. Increased glutamate dehydrogenase activity is responsible for hyperammonaemia, an increased alpha ketoglutarate level and, consequently, increased Krebs cycle activity and B-cell ATP/ADP ratio, with subsequent exaggerated insulin release. Sulphonylureas, such as tolbutamide, stimulate insulin secretion by binding directly to SURs. 52 P. de Lonlay et al. Figure 1 Insulin metabolism. Several pathways are involved in the regulation of glucose, including insulin secretion by pancreatic B-cells, explaining the modality of effectiveness of medical treatments as diazoxide, somatostatin and a protein-restricted diet. Glucose and other fuels, such as amino acids, stimulate insulin secretion through their metabolism to raise the intracellular ATP/ADP ratio. Glucokinase, the enzyme that initiates B-cell glucose metabolism, has a high Km for glucose, and, thus, the circulating concentrations of glucose directly determine the rate of glucose oxidation and subsequent release of insulin. Increases in the ATP/ADP ratio activate a plasma membrane protein, the sulphonylurea receptor (SUR), to cause closure of a potassium channel (KATP channel). This, in turn, leads to depolarization of the membrane, an influx of extracellular calcium, and the release of insulin from storage granules. Leucine, one of the most potent amino acids in stimulating insulin secretion, acts indirectly as a positive allosteric affector of glutamate dehydrogenase to increase the rate of oxidation of glutamate. Increased glutamate dehydrogenase activity is responsible for increased alpha ketoglutarate level, increased Krebs cycle activity and B-cell ATP/ADP ratio, and subsequently leads to exaggerated insulin release. Diazoxide inhibits insulin secretion by binding to SURs. Insulin secretion in response to tolbutamide has recently been suggested to separate focal forms (tolbutamide responsive) from diffuse forms (tolbutamide insensitive). Tolbutamide triggers insulin secretion in focal forms, but not in diffuse forms. This hypothesis has not been confirmed in our experience (submitted for publication). Patients who are treated surgically have to be classified according to histological criteria.9–11 The focal form is defined as a focal adenomatous hyperplasia. The lesion measures 2.5–7.5 mm in diameter, differing from true adult-type pancreatic adenoma which is clearly limited with a different topographic distribution. Diffuse CI shows abnormal B-cell nuclei in all sections of the pancreas. In the absence of any distinctive clinical feature, and because pre-operative classical radiology of the pancreas, including echotomography, computed tomography scan and nuclear magnetic resonance, is not efficient to screen the focal forms, pancreatic venous catheterization12,13 and pancreatic arteriography are the only pre-operative proce- dures currently available for locating the site of insulin secretion. They are not performed before the age of 1 month, to exclude patients with transient forms, or in patients with hyperammonaemia who are likely to have diffuse hyperinsulinism. The estimated incidence of CI is 1/50 000 live births, but the incidence may be high as 1/2500 in countries with substantial inbreeding. The two histological forms that can be found in both neonatal and infancy-onset hyperinsulinism correspond with distinct molecular entities.12,13. Focal islet-cell hyperplasia is associated with hemi- or homozygosity of a paternally inherited mutation of the SUR1 or the inward-rectifying potassium channel (Kir6.2) genes on chromosome 11p15 and loss of the maternal allele in the hyperplastic islets neonates.14,15 The loss of the 11p15 maternal allele leads to an unbalanced expression of 11p15 imprinted genes, namely growth factor and suppressor tumour genes. Focal lesion is probably a sporadic event, as suggested by the somatic molecular abnormality in the pancreas and by discordant identical twins. However, the co-existence of a focal and a diffuse lesion in the same family cannot Neonatal hypoglycaemia: aetiologies be excluded. Diffuse hyperinsulinism is a heterogeneous disorder involving the genes encoding the SUR or the inward-rectifying potassium channel7,16–19 in recessively inherited hyperinsulinism, or more rarely, the glucokinase gene20 or other loci21 in dominantly inherited hyperinsulinism, and the glutamate dehydrogenase gene when hyperammonaemia is associated with hyperinsulinism.22,23 In this case, transmission can be sporadic or dominant. More recently, short-chain l-3-hydroxyacylCoA dehydrogenase (SCHAD) has been implicated.24 The clinical presentation of hyperinsulinism related to potassium channels (with focal or diffuse lesion) depends on the age of onset of hypoglycaemia. In contrast, hyperinsulinism associated with hyperammonaemia is less severe, even when the onset is neonatal. Patients with neonatal CI who are responsive to diazoxide may have a transient form of hyperinsulinism or the hyperinsulinism– hyperammonaemia syndrome. Adenoma differs radically from focal CI by the late onset of hypoglycaemia and the histological lesion. To date, its aetiology is unknown apart from adenoma related to the MEN1 syndrome with dominant mutation on the MEN1 gene following menine protein deficiency, a loss of the 11p13 region and Bourneville's tuberous sclerosis. Finally, many genes could be implicated, namely all the genes playing a role in insulin secretion. The differential diagnosis is Munchausen by proxy.25 Fatty acid oxidation and ketogenesis disorders Since the first description of fasting hypoglycaemia revealing medium-chain acylCoA dehydrogenase and hepatic carnitine palmitoyl transferase deficiency,26 defects of FAO have become one of the most important causes of genetic hypoglycaemia.27 In a series of 107 patients with FAO defects, hypoketotic hypoglycaemia was observed in 60% of cases and was the revealing symptom in about 50%. In most cases, the inappropriately low levels of plasma ketones were suggested by negative acetest in urine at the time of acute attack, and then further demonstrated by carefully supervised fasting test.28 In neonates, hypoglycaemia was observed within the first 72 h of life in term eutrophic neonates presenting with hypotonia, poor feeding and life-threatening events such as apnea, collapse, dyspnoea or seizures. In almost all cases, hypoglycaemia was associated with cardiac symptoms (arrhythmia, conduction defects, cardiomyopathy),27 moderate hepatomegaly, metabolic acidosis, hyperlactacidaemia, hyperammonaemia, 53 and moderate elevation of transaminases. The glucose requirement to maintain blood glucose within normal limits was normal. In neonates, hypoglycaemia never raised a real diagnostic problem. In infancy, by contrast, hypoglycaemia usually appeared as an isolated revealing sign in the context of fasting or catabolism. Diagnosis of hyperinsulinism, hypopituitarism and fructose biphosphatase deficiency were frequently considered and ruled out before reaching the correct diagnosis of FAO disorder. A moderate hepatomegaly was very frequent during an acute attack with negative, inappropriately low urine test for ketones, mild acidosis, mild hyperlactacidaemia, mild hyperammonaemia and moderately increased serum transaminases. As a whole, hypoglycaemic attacks revealing an FAO disorder in infancy presented either like a fasting hypoglycaemia with no ketonuria, or resembled Reye's syndrome. In our series, there were no clinical, biological or histological criteria which allowed us to separate a priori FAO disorders from idiopathic Reye's syndrome. All kinds of long- and medium-chain fatty acid disorders can present with Reye's syndrome, hepatomegaly, steatosis and hypoketotic hypoglycaemia. In these defects, hypoglycaemia results from underproduction of glucose by the liver associated with above-normal consumption of glucose by peripheral tissues due to the incapacity of these tissues to oxidize fatty acids and to the absence of ketone bodies as alternative fuels. Both phenomena can be quickly reversed in long-chain FAO disorders by giving medium-chain triglycerides which dramatically increase blood glucose and ketone body concentrations. The diagnostic approach to FAO disorders is mainly based on blood and urine investigations performed during acute metabolic stress. Investigation of FAO disorders has recently been simplified radically by investigating acylcarnitine profiles by FAB-MSMS or electrospray MSMS from simple blood spots collected on a Guthrie card.29 In addition to this very efficient technique, the determination of urinary organic acid profiles by classical GC–MS can give highly specific patterns or suggestive but non-specific patterns (like saturated and unsaturated medium- and long-chain dicarboxylic acids). However, a normal pattern does not allow the exclusion of an FAO disorder.30 If no material is collected at the time of acute attack or if the results are incomplete or ambiguous, a function test that challenges the metabolic pathway may provide a tentative diagnosis. When performing such a function test, it is very important to adhere to a strictly defined protocol to obtain the maximum diagnostic information and minimize the risk 54 of metabolic complications. Two tests are very informative but dangerous; the fasting test and the long-chain triglycerides loading test. Finally, inherited defects of FAO can be diagnosed by in vitro testing using fresh circulating lymphocytes or intact cultured fibroblasts to oxidize individual fatty acids. To date, all the described defects involving the liver have been expressed in these cells.27 HMGCoA lyase and HMGCoA synthetase deficiencies have a similar presentation to FAO disorders and can be approached using the same procedures.31–35 However, they require specific enzymatic determinations for definitive confirmation. Disturbances in the synthesis and degradation of glycogen and gluconeogenesis defects The most frequent glycogen anomaly leading to hypoglycaemia is glycogenosis type I.36 Two very rare enzyme defects disturb the synthesis of liver glycogen; glycogen synthase deficiency and branching enzyme deficiency or glycogen storage disease type IV. The latter presents mainly with cirrhosis and not with preponderant hypoglycaemia. In glycogen synthase deficiency,37 the synthesis of liver glycogen is profoundly, although not completely, impaired. The defect provokes a severe morning hypoglycaemia with a very characteristic profile that appears in early infancy with cessation of nocturnal feeding. After an overnight fast (second phase of glucose homeostasis), there is a severe hypoglycaemia with hyperketonaemia, low lactate and low alanine. Conversely, hyperglycaemia and hyperlactataemia occur after meals, while ketonaemia decreases to a normal value. Glucagon causes a rise in glucose 3 h after a meal with a fall in lactate and alanine, but no effect of glucagon is seen after a 12-h fast. The enzyme defect can only be demonstrated in the liver, and not in other tissues. Several enzyme defects in the glycogenolytic pathway impair the degradation of liver glycogen, and present with a profound hypoglycaemia which also appears in the second phase of glucose homeostasis. In glucose-6-phosphatase deficiency (glycogen storage disease type Ia) and glucose-6phosphate translocase (glycogenosis type Ib), profound hypoglycaemia generally appears 2.5–3.5 h after a meal because the enzyme defect not only suppresses the release of glucose from glycogen but also the formation of glucose by the gluconeogenic pathway. Diagnosis is easy, based upon the large hepatomegaly, lactic acidosis, slight ketosis and hyperuricaemia. In glycogenosis type Ib, fluctuant P. de Lonlay et al. neutropenia responsible for recurrent infections is a near-constant finding. Confirmation of diagnosis is now based directly upon molecular investigation of the glucose-6-phosphatase gene36 or of the glucose-6-phosphate translocase gene. This molecular diagnosis avoids performing a liver biopsy for enzymatic assay. In amylo-1,6-glucosidase deficiency (glycogen storage disease type III), hypoglycaemia is usually mild compared with glycogenoses type I because degradation of glycogen by phosphorylase remains possible and the gluconeogenic pathway is intact. In this disorder, a protuberant abdomen with an enormous hepatomegaly is the striking feature. There is no fasting lactic acidosis and frank ketonuria is associated with fasting hypoglycaemia. A moderate elevation of lactate concentration can be observed in the postabsorptive state. A mild elevation of creatine kinase reflects associated muscular glycogenosis. Cardiomyopathy is present in some molecular subtypes. Hypoglycaemia is very rare in phosphorylase complex deficiency (phosphorylase itself and phosphorylase-b-kinase = glycogenosis type VI and IX). Hypoglycaemia does not exist in glycogenosis type II (alpha-glucosidase deficiency). In addition to glucose-6-phosphatase deficiency, several enzyme defects disturb the formation of glucose by the gluconeogenic pathway,38 namely the deficiencies of fructose-1,6-biphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Hypoglycaemia is a major feature in fructose-1,6-biphosphatase deficiency. It usually strikes after an overnight fast or during catabolic states at the end of phase 2 or the beginning of phase 3 of glucose homeostasis. Moderate hepatomegaly is frequent during an acute attack but does not always occur. Hypoglycaemia is associated with moderate hyperketonaemia and lactic acidosis which can be preponderant. Diagnosis relies upon a fasting test performed under careful supervision that displays a characteristic profile with a progressive decrease of glucose concentrations contrasting with a progressive increase of lactate concentrations. These patients are fructose intolerant; acute fructose administration lead to acute hypoglycaemia, as in fructose-1-phosphate aldolase deficiency. Diagnosis relies on the measurement of fructose biphosphatase activity in lymphocytes or on liver biopsy. To date, there is no well-documented case of phosphoenolpyruvate carboxykinase deficiency presenting with recurrent hypoglycaemia. Hypoglycaemia is also only occasionally mentioned in pyruvate carboxylase deficiency, probably because this defect does not Neonatal hypoglycaemia: aetiologies affect the entrance of a number of substrates, such as glycerol and serine, into the gluconeogenic pathway.4 Other rare enzymatic or transporter disorders Ketolytic defects Ketolytic defects (succinylCoA: acetoacetate transferase and acetoacetylCoA thiolase deficiencies) rarely present with recurrent attacks of hypoglycaemia.31–35 Mostly, hypoglycaemia strikes in the third phase of glucose homeostasis, and is associated with severe ketoacidosis, low lactate and low alanine. Diagnosis relies upon the demonstration of a permanent ketosis during a nycthemeral cycle, and on unusually high concentrations of ketone bodies compared with free fatty acids. Enzymatic assays (not performed routinely) can be done on cultured fibroblasts. These cases, like those with glycogen synthetase deficiency, could be easily mistaken for ketotic hypoglycaemia syndrome if functional tests, including a 24-h cycle and fasting test, are not performed in a well-trained metabolic ward.33 We recently observed two patients affected with complex IV deficiency and one patient with complex CIII deficiency of the respiratory chain, who presented with isolated recurrent attacks of fasting hypoglycaemia mimicking fructose-1,6-diphosphatase deficiency (with moderate hyperketosis and hyperlactacidaemia) (J.M. Saudubray, unpublished observations). Phosphomannose isomerase deficiency Phosphomannose isomerase deficiency is responsible for congenital glycosylation disorder (CDG) type Ib. Less frequently, phosphomannomutase deficiency (CDG Ia) can lead to predominant hypoglycaemia due to hyperinsulinism. Glucose transporter deficiencies The pathophysiology of Fanconi Bickel syndrome (FBS) was identified recently.39,40 FBS is a rare well-defined clinical entity which is inherited in an autosomal-recessive mode. It is characterized by hepatorenal glycogen accumulation, fasting hypoglycaemia, postprandial hyperglycaemia and hypergalactosaemia (indicating an impaired utilization of these two monosaccharides), and proximal renal tubular dysfunction. In contrast to other types of glycogen storage diseases caused by enzymatic defects of glycogenolysis, FBS has recently 55 been shown to result from a defective monosaccharide transporter, GLUT2, in cell membranes of different tissues.40 It thus represents the first disease with hypoglycaemia caused by a defect of a member of the facilitative glucose transporter family. The diagnosis of this disorder, easily suspected on the very suggestive clinical pattern, relies upon molecular investigation of the GLUT2 gene.40 The GLUT1 deficiency syndrome caused by haplo-insufficiency of the blood–brain barrier hexose carrier described recently41 belongs to the same family of glucose transporter disorders. However, in this disorder, the low glucose concentration is only demonstrable in cerebrospinal fluid (CSF) while simultaneous blood glucose concentration is normal. The disorder presents with severe infantile seizures with delayed development and acquired microcephaly. The diagnosis is suspected on persistent hypoglycorrachia (low CSF glucose) with low to normal lactate, and normal circulating simultaneous blood glucose. Confirmation can be made by the demonstration of diminished transport of hexose into isolated red blood cells and mutation analyses of the GLUT1 gene. Genetic endocrine disorders Hypoglycaemia due to GH deficiency in the newborn has been recognized for many years, with symptomatic hypoglycaemia as a revealing symptom.42,43 In GH deficiency, hypoglycaemia is rather rare (less than 20%). Symptomatic hypoglycaemia is more frequent in lean children below 4 years of age. If the deficiency is due to defects early in development, such as GH gene deletion or the rare instances of mutations in the GH-releasing hormone (GHRH) receptor,44–46 micropenis is also found in males.43 As these characteristics are also found in Laron's syndrome, the early deficiency of male hormone expression in utero seems to be related to the absence of GH action and/or IGF1 deficit.47 Similar findings can be observed in malformations of the hypothalamic area, leading to multiple pituitary hormone deficiencies, in which case the microphallus and hypogonadism are due to a combination of primary gonadotrophin deficiency and lack of GH, leading to secondary deficiency of IGF1. In all these patients, hypoglycaemia occurs during fasting (second and third phase of glucose homeostasis); ketone body concentrations are variable—low in neonates and young infants, and high in children. Hypoglycaemia responsiveness to glucagon is also variable—mild, absent or dramatic—the latter response being similar to that observed in hyperinsulinism. 56 Laron's syndrome is due to primary IGF1 deficiency (molecular defects of the GH receptor, in the postreceptor area or in the synthesis and action of IGF1). Patients with this syndrome are indistinguishable from patients with isolated GH deficiency, but have excessively high serum GH levels which are ineffective (GH resistance, insensitivity). Marked hypoglycaemia in addition to dwarfism and obesity is one of the characteristics of these patients. Hypoglycaemia is symptomatic in infancy and becomes asymptomatic with advancing age. Before 6 years of age, children with Laron's syndrome present with insulin hypoglycaemia nonresponsiveness denoting a defect in the counterregulatory mechanism. These rare syndromes raise the interesting question of the interaction between GH and IGF1 activities on glucose metabolism. The mechanism by which IGF1 deprivation induces hypoglycaemia is probably an interplay between the role that IGF1 has on glucose transport, phosphorylation, glycolysis and glycogen synthesis, and the lack of GH-induced hepatic gluconeogenesis. Hypoglycaemia associated with isolated ACTH deficiency is as rare as hypoglycaemia due to primary adrenal failure, such as in adrenoleukodystrophy. Glucagon deficiency can also be associated with hypoglycaemia.48 Conclusions In our experience, most genetic causes of hypoglycaemia are easy to elucidate. Diagnosis relies upon a limited number of parameters, mostly clinical, uncomplicated tests. Commemorative and clinical features are very important (age at onset, severity and frequency of hypoglycaemic attacks, timing of hypoglycaemia, glucose requirement to maintain normal blood glucose concentrations, ketosis, glucagon responsiveness, hepatomegaly, short stature, dysmorphy). Knowledge of the homeostatic mechanisms which maintain normal blood glucose in early life is crucial to the diagnosis. Hypoglycaemia in the absorptive phase is very likely to be due to hyperinsulinism. Children presenting acutely with hypoglycaemia are often dangerously ill and possibly moribund. In such an emergency, a detailed history and examination may have to be differed. Too often, blood is only taken for glucose and possible electrolytes before glucose is given. When the child has recovered, it may be very difficult to establish a diagnosis without recourse to a fasting test with the associated risks. It is extremely important to collect appropriate samples at the time of the acute event. This will provide a diagnosis or at least indicate which group P. de Lonlay et al. of disorders is likely. Blood should always be taken for glucose, insulin, GH, cortisol, lactate, amino acids with alanine quantification if possible, 3-hydroxybutyrate, free fatty acids, Guthrie card for acylcarnitine profile, and liver function test. The first urine pass must always be tested for ketones, allowing a first simple separation between ketotic and non-ketotic hypoglycaemia, and saved for organic acids analysis. Practice points Clinical features to note • • • • Timing of hypoglycaemia (fasting or feeding) Hepatomegaly Severity of hypoglycaemia Quantity of glucose infusion to normalize glycaemia • Head circumference • Other associated symptoms Measurements during hypoglycaemia • • • • • • • • • • Glycaemia Lactate NH3 Ketone bodies Liver function Insulin, GH, insulin-like growth factor 1, cortisol, c peptide Amino acids (plasma) Organic acids (urine) Acylcarnitine (plasma) Glucagon test if feeding hypoglycaemia (not for fasting hypoglycaemia) If symptoms: transferrin Western blot (congenital disorders of glycosylation). Research directions • New syndromes are known to be associated with hypoglycaemia, namely Sotos syndrome and congenital disorders of glycosylation. • New genes will be found to be implicated in hypoglycaemia, i.e. all the genes playing a role in glycaemia regulation. • Animal models should be researched, i.e. mice with hyperinsulinism. • All mechanisms are not yet understood. 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