Charles A. Stanley

A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene.

Type 1 diabetes (T1D) in children results from autoimmune destruction of pancreatic beta cells, leading to insufficient production of insulin. A number of genetic determinants of T1D have already been established through candidate gene studies, primarily within the major histocompatibility complex but also within other loci. To identify new genetic factors that increase the risk of T1D, we performed a genome-wide association study in a large paediatric cohort of European descent. In addition to confirming previously identified loci, we found that T1D was significantly associated with variation within a 233-kb linkage disequilibrium block on chromosome 16p13. This region contains KIAA0350, the gene product of which is predicted to be a sugar-binding, C-type lectin. Three common non-coding variants of the gene (rs2903692, rs725613 and rs17673553) in strong linkage disequilibrium reached genome-wide significance for association with T1D. A subsequent transmission disequilibrium test replication study in an independent cohort confirmed the association. These results indicate that KIAA0350 might be involved in the pathogenesis of T1D and demonstrate the utility of the genome-wide association approach in the identification of previously unsuspected genetic determinants of complex traits.

Primary Carnitine Deficiency Due to a Failure of Carnitine Transport in Kidney, Muscle, and Fibroblasts

CARNITINE (β-hydroxy-γ-trimethylaminobutyric acid) is an essential cofactor for the oxidation of fatty acids by mitochondria. It serves to carry long-chain fatty acids in the form of their acyl-car...

A Nonsense Mutation in the Inward Rectifier Potassium Channel Gene, Kir6.2, Is Associated With Familial Hyperinsulinism

ATP-sensitive potassium (KATP) channels are an essential component of glucose-dependent insulin secretion in pancreatic islet β-cells. These channels comprise the sulfonylurea receptor (SUR1) and Kir6.2, a member of the inward rectifier K+ channel family. Mutations in the SUR1 subunit are associated with familial hyperinsulinism (HI) (MIM:256450), an inherited disorder characterized by hyperinsulinism in the neonate. Since the Kir6.2 gene maps to human chromosome 11p15.1 (1,2), which also encompasses a locus for HI, we screened the Kir6.2 gene for the presence of mutations in 78 HI probands by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses. A nonsense mutation, Tyr→Stop at codon 12 (designated Y12X) was observed in the homozygous state in a single proband. 86Rb+ efflux measurements and single-channel recordings of COS-1 cells co-expressing SUR1 and either wild-type or Y12X mutant Kir6.2 proteins confirmed that KATP channel activity was abolished by this nonsense mutation. The identification of an HI patient homozygous for the Kir6.2/Y12X allele affords an opportunity to observe clinical features associated with mutations resulting in an absence of Kir6.2. These data provide evidence that mutations in the Kir6.2 sub-unit of the islet β-cell KATP channel are associated with the HI phenotype and also suggest that the majority of HI cases are not attributable to mutations in the coding region of the Kir6.2 gene.

Medium-chain acyl-CoA dehydrogenase deficiency. Diagnosis by stable-isotope dilution measurement of urinary n-hexanoylglycine and 3-phenylpropionylglycine.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, one of the most common inherited metabolic disorders, is often mistaken for the sudden infant death syndrome or Reyes syndrome. Diagnosing it has been difficult because of a lack of fast and reliable diagnostic methods. We developed a stable-isotope dilution method to measure urinary n-hexanoylglycine, 3-phenylpropionylglycine, and suberylglycine, and we retrospectively tested its accuracy in diagnosing MCAD deficiency. We measured the concentrations of these three acylglycines in 54 urine samples from 21 patients with confirmed MCAD deficiency during the acute and asymptomatic phases of the illness and compared the results with the concentrations in 98 samples from healthy controls and patient controls with various diseases. The levels of urinary hexanoylglycine and phenylpropionylglycine were significantly increased in all samples from the patients with MCAD deficiency, clearly distinguishing them from both groups of controls. Although urinary suberylglycine was increased in the patients, the range of values in the normal controls who were receiving formula containing medium-chain triglycerides was very wide, overlapping somewhat with the values in the patients with asymptomatic MCAD deficiency. These results indicate that the measurement of urinary hexanoylglycine and phenylpropionylglycine by our method is highly specific for the diagnosis of MCAD deficiency. The method is fast and can be applied to random urine specimens, without any pretreatment of patients.Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, one of the most common inherited metabolic disorders, is often mistaken for the sudden infant death syndrome or Reyes syndrome. Diagnosing it has been difficult because of a lack of fast and reliable diagnostic methods. We developed a stable-isotope dilution method to measure urinary n-hexanoylglycine, 3-phenylpropionylglycine, and suberylglycine, and we retrospectively tested its accuracy in diagnosing MCAD deficiency. We measured the concentrations of these three acylglycines in 54 urine samples from 21 patients with confirmed MCAD deficiency during the acute and asymptomatic phases of the illness and compared the results with the concentrations in 98 samples from healthy controls and patient controls with various diseases. The levels of urinary hexanoylglycine and phenylpropionylglycine were significantly increased in all samples from the patients with MCAD deficiency, clearly distinguishing them from both groups of controls. Although urinary suberylglycine was increased in the patients, the range of values in the normal controls who were receiving formula containing medium-chain triglycerides was very wide, overlapping somewhat with the values in the patients with asymptomatic MCAD deficiency. These results indicate that the measurement of urinary hexanoylglycine and phenylpropionylglycine by our method is highly specific for the diagnosis of MCAD deficiency. The method is fast and can be applied to random urine specimens, without any pretreatment of patients.

Effect of Tri-Iodothyronine Replacement on the Metabolic and Pituitary Responses to Starvation

To determine the implication of decreased T3 production during fasting, seven normal men were fasted for 80 hours on two occasions; they received 5 microgram of T3 every three hours durnig the second fast. The mean serum T3 concentration declined during the control fast from 120 to 73 ng per deciliter (P less than 0.01), but remained slightly above base-line values during the T3 fast. Mean serum T4 concentrations did not change, and mean serum rT3 concentrations increased, during both fasts. The peak serum TSH increment after TRH was 11.1 micromicron per milliliter before fasting, 8.9 (not significant) after the control fast and 2.2 (P less than 0.01) after the T3 fast. Urea excretion was 9.1 per cent higher during the T3 fast; there were no differences in the changes in blood glucose, plasma fatty acids or other substrates during the two fasts. Pretreatment with potassium iodide lowered serum T4 concentrations and increased the serum TSH response to TRH after fasting. We conclude that the decrease in serum T3 concentrations during fasting spares muscle protein. Fasting is accompanied by a lower set point of TSH secretion, which remains sensitive to changes in serum thyroid hormone concentrations.

Hyperinsulinism in infants and children.

Hyperinsulinism is the most common cause of hypoglycemia in early infancy. Congenital hyperinsulinism, formerly termed nesidioblastosis, is usually caused by genetic defects in beta-cell regulation, including a severe recessive disorder of the sulfonylurea receptor, a milder dominant form of hyperinsulinism, and a syndrome of hyperinsulinism plus hyperammonemia. Transient neonatal hyperinsulinism may be associated with perinatal asphyxia or small-for-dates birthweight and maternal diabetes. To prevent permanent brain damage from hypoglycemia, the treatment of infants with hyperinsulinism must be prompt and aggressive. A combination of medical therapy with diazoxide or octreotide, a long-acting somatostatin analog, and surgical 95% subtotal pancreatectomy may be required.

Long-chain acyl coenzyme A dehydrogenase deficiency : an inherited cause of nonketotic hypoglycemia

ABSTRACT: Three children from unrelated families presented in early childhood with hypoglycemia and cardiorespiratory arrests associated with fasting. Significant hepatomegaly, cardiomegaly, and hypotonia were present at the time of initial presentation. Ketones were not present in the urine at the time of hypoglycemia in any patient; however, dicarboxylic aciduria was documented in one patient at the time of the acute episode and in two patients during fasting studies. Total plasma carnitine concentration was low with an increased esterified carnitine fraction. These findings suggested a defect in mitochondrial fatty acid oxidation, and specific assays were performed for the acyl coenzyme A (CoA) dehydrogenases. These analyses showed that the activity of the long-chain acyl CoA dehydrogenase was less than 10% of control values in fibroblasts, leukocytes, and liver tissue. Activities of the medium-chain, short-chain, and isovaleryl CoA dehydrogenases were not different from control values. With cultured fibroblasts, CO2 evolution from long-chain fatty acids was significantly reduced, while CO2 evolution from medium-chain and short-chain fatty acids was comparable to control values—findings consistent with a defect early in the β-oxidation sequence. Studies of acyl CoA dehydrogenase activities in fibroblasts and leukocytes from parents of the patients showed levels of long-chain acyl CoA dehydrogenase activity intermediate between affected and control values and indicated an autosomal recessive form of inheritance of this enzymatic defect. These results describe a previously unrecognized metabolic disorder of fatty acid oxidation due to a deficiency of the long-chain acyl CoA dehydrogenase which may present in early childhood with disastrous consequences. This diagnosis should be considered in children who present with nonketotic hypoglycemia, carnitine insufficiency, and inadequately explained cardiorespiratory arrests.

Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels.

Summary: Three children in two families presented in early childhood with episodes of illness associated with fasting which resembled Reyes syndrome: coma, hypoglycemia, hyperammonemia, and fatty liver. One child died with cerebral edema during an episode. Clinical studies revealed an absence of ketosis on fasting (plasma beta-hydroxybutyrate < 0.4 mmole/liter) despite elevated levels of free fatty acids (2.6–4.2 mmole/liter) which suggested that hepatic fatty acid oxidation was impaired. Urinary dicarboxylic acids were elevated during illness or fasting. Total carnitine levels were low in plasma (18–25 μmole/liter), liver (200–500 nmole/g), and muscle (500–800 nmole/g); however, treatment with L-carnitine failed to correct the defect in ketogenesis. Studies on ketone production from fatty acid substrates by liver tissue in vitro showed normal rates from short-chain fatty acids, but very low rates from all medium and long-chain fatty acid substrates. These results suggested that the defect was in the mid-portion of the intramitochondrial beta-oxidation pathway at the medium- chain acyl-CoA dehydrogenase step. A new assay for the electron transfer flavoprotein-linked acyl-CoA dehydrogenases was used to test this hypothesis. This assay follows the decrease in electron transfer flavoprotein fluorescence as it is reduced by acyl-CoA-acyl-CoA dehydrogenase complex. Results with octanoyl-CoA as substrate indicated that patients had less than 2.5% normal activity of medium-chain acyl-CoA dehydrogenase. The activities of short-chain and isovaleryl acyl-CoA dehydrogenases were normal; the activity of long-chain acyl-CoA dehydrogenase was one-third normal.These results define a previously unrecognized inherited metabolic disorder of fatty acid oxidation due to deficiency of medium-chain acyl-CoA dehydrogenase. The carnitine deficiency in these patients appears to be a secondary consequence of their defect in fatty acid oxidation. It is possible that other patients with “systemic carnitine deficiency,” who fail to respond to carnitine therapy, may also have defects in fatty acid oxidation similar to medium-chain acyl-CoA dehydrogenase deficiency.

Carnitine Deficiency Disorders in Children

Abstract: Mitochondrial oxidation of long‐chain fatty acids provides an important source of energy for the heart as well as for skeletal muscle during prolonged aerobic work and for hepatic ketogenesis during long‐term fasting. The carnitine shuttle is responsible for transferring long‐chain fatty acids across the barrier of the inner mitochondrial membrane to gain access to the enzymes of β‐oxidation. The shuttle consists of three enzymes (carnitine palmitoyltransferase 1, carnitine acylcarnitine translocase, carnitine palmitoyl‐transferase 2) and a small, soluble molecule, carnitine, to transport fatty acids as their long‐chain fatty acylcarnitine esters. Carnitine is provided in the diet (animal protein) and also synthesized at low rates from trimethyl‐lysine residues generated during protein catabolism. Carnitine turnover rates (300‐500 μmol/day) are <1% of body stores; 98% of carnitine stores are intracellular (total carnitine levels are 40‐50 μM in plasma vs. 2‐3 mM in tissue). Carnitine is removed by urinary excretion after reabsorption of 98% of the filtered load; the renal carnitine threshold determines plasma concentrations and total body carnitine stores. Because of its key role in fatty acid oxidation, there has long been interest in the possibility that carnitine might be of benefit in genetic or acquired disorders of energy production to improve fatty acid oxidation, to remove accumulated toxic fatty acyl‐CoA metabolites, or to restore the balance between free and acyl‐CoA. Two disorders have been described in children where the supply of carnitine becomes limiting for fatty acid oxidation: (1) A recessive defect of the muscle/kidney sodium‐dependent, plasma membrane carnitine symporter, which presents in infancy with cardiomyopathy or hypoketotic hypoglycemia; treatment with oral carnitine is required for survival. (2) Chronic administration of pivalate‐conjugated antibiotics in which excretion of pivaloyl‐carnitine can lead to carnitine depletion; tissue levels may become low enough to limit fatty acid oxidation, although no cases of illness due to carnitine deficiency have been described. There is speculation that carnitine supplements might be beneficial in other settings (such as genetic acyl‐CoA oxidation defects—“secondary carnitine deficiency”, chronic ischemia, hyperalimentation, nutritional carnitine deficiency), but efficacy has not been documented. The formation of abnormal acylcarnitines has been helpful in expanded newborn screening programs using tandem mass‐spectrometry of blood spot acylcarnitine profiles to detect genetic fatty acid oxidation defects in neonates. Carnitine‐deficient diets (vegetarian) do not have much effect on carnitine pools in adults. A modest 50% reduction in carnitine levels is associated with hyperalimentation in newborn infants, but is of doubtful significance. The above considerations indicate that carnitine does not become rate‐limiting unless extremely low; testing the benefits of nutritional supplements may require invasive endurance studies of fasting ketogenesis or muscle and cardiovascular work.

Mechanisms of Disease: advances in diagnosis and treatment of hyperinsulinism in neonates

Hyperinsulinism is the single most common mechanism of hypoglycemia in neonates. Dysregulated insulin secretion is responsible for the transient and prolonged forms of neonatal hypoglycemia, and congenital genetic disorders of insulin regulation represent the most common of the permanent disorders of hypoglycemia. Mutations in at least five genes have been associated with congenital hyperinsulinism: they encode glucokinase, glutamate dehydrogenase, the mitochondrial enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase, and the two components (sulfonylurea receptor 1 and potassium inward rectifying channel, subfamily J, member 11) of the ATP-sensitive potassium channels (KATP channels). KATP hyperinsulinism is the most common and severe form of congenital hyperinsulinism. Infants suffering from KATP hyperinsulinism present shortly after birth with severe and persistent hypoglycemia, and the majority are unresponsive to medical therapy, thus requiring pancreatectomy. In up to 40–60% of the children with KATP hyperinsulinism, the defect is limited to a focal lesion in the pancreas. In these children, local resection results in cure with avoidance of the complications inherent to a near-total pancreatectomy. Hyperinsulinism can also be part of other disorders such as Beckwith–Wiedemann syndrome and congenital disorders of glycosylation. The diagnosis and management of children with congenital hyperinsulinism requires a multidisciplinary approach to achieve the goal of therapy: prevention of permanent brain damage due to recurrent hypoglycemia.