Oliver Blankenstein

Proopiomelanocortin Deficiency Treated with a Melanocortin-4 Receptor Agonist

Patients with rare defects in the gene encoding proopiomelanocortin (POMC) have extreme early-onset obesity, hyperphagia, hypopigmentation, and hypocortisolism, resulting from the lack of the proopiomelanocortin-derived peptides melanocyte-stimulating hormone and corticotropin. In such patients, adrenal insufficiency must be treated with hydrocortisone early in life. No effective pharmacologic treatments have been available for the hyperphagia and obesity that characterize the condition. In this investigator-initiated, open-label study, two patients with proopiomelanocortin deficiency were treated with setmelanotide, a new melanocortin-4 receptor agonist. The patients had a sustainable reduction in hunger and substantial weight loss (51.0 kg after 42 weeks in Patient 1 and 20.5 kg after 12 weeks in Patient 2).

Proposal for a standardized protocol for 18F-DOPA-PET (PET/CT) in congenital hyperinsulinism

a Department of Pediatrics and Neonatology, Otto von Guericke University Magdeburg, Magdeburg , and b Department of Pediatric Endocrinology, Charité University of Medicine Berlin, Berlin , Germany; c Department of Pediatrics, Odense University Hospital, Odense , Denmark; d Department of Pediatrics, Hôpital Necker Enfants Malades, Paris , France; e London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children, London , UK; f Department of Nuclear Medicine/PET Centre, University Medical Centre Groningen, Groningen , The Netherlands; g Department of Oncology and Radiotherapy, Turku University Central Hospital, Turku , Finland; h PET Centre Berlin, Berlin , Germany; i Department of Pediatrics, King Abdilaziz Medical City, Riyadh , Saudi Arabia; j University Children’s Hospital for Pediatric Endocrinology and Diabetology, Helsinki , Finland; k Department of Pathology, Cliniques Universitaires St Luc, UCL, Brussels , Belgium; l CEA Service Hospitalier Frédéric Joliot, Orsay , France; m Department of Pediatrics, University of Zurich, Zurich , Switzerland, and n Department of Pediatric Surgery, Hôpital Necker Enfants Malades, Paris , France

Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor

An internal fragment of the cfb gene from group B streptococcal (GBS) strain R268 was amplified by polymerase chain reaction (PCR) using degenerate primers with sequences derived from the CAMP-factor amino acid (aa) sequence of GBS strain NCTC8181 [Rühlmann et al. (1988) FEBS Lett 235: 262–266]. After cloning and sequencing this fragment, the remainder of cfb and the adjacent 5′ and 3′ sequences were amplified by inverted PCR of genomic DNA and directly sequenced from the PCR product. Within the 1560 bp sequenced, a complete cfb gene deviating in two deduced aa residues from the published sequence was identified. In addition, the cfbR268 sequence contained a 29-aa leader peptide. Using primers directed to the 5′ and 3′ ends of cfb for PCR, a cfb gene of uniform size could be detected in 19 clinical GBS isolates including three phenotypically CAMP-negative strains. Utilizing Northern blot analysis and primer extension assays, the cfbR268 promoter was located and the length of the cfb transcript was assessed at about 1100 bp. In a parallel experiment, no cfb transcript could be detected from the CAMP-negative GBS strain 74–360. The complete cfbR268 gene and different portions of its 5′ and 3′ ends were cloned into the plasmid pJLA602 and expressed in E. coli DH5α. The recombinant peptides could be detected by Western immunoblots with polyclonal antiserum. Only the full-sized recombinant CAMP-factor was found to exert co-hemolytic activity in a sheep-blood agar assay. This co-hemolytic activity could be inhibited by anti-CAMP antiserum.

Hyperinsulinaemic hypoglycaemia: biochemical basis and the importance of maintaining normoglycaemia during management

In patients with suspected hyperinsulinaemic hypoglycaemia, blood glucose concentrations should be maintained within the normal range during routine management Hyperinsulinaemic hypoglycaemia (HH) is a major cause of recurrent and persistent hypoglycaemia in infancy and childhood.1 Rapid diagnosis, avoidance of recurrent and repeated episodes of hypoglycaemia and prompt management of the hypoglycaemia are vital in preventing brain damage and mental retardation.2 Unfortunately, a large proportion of children with HH still develop brain damage as a consequence of delayed diagnosis and subsequent management. HH can be either congenital or secondary to certain risk factors (such as intrauterine growth retardation). Congenital hyperinsulinism involves either defects in the genes ABCC8 and KCNJ11 (encoding for the two proteins SUR1 and KIR6.2 of the pancreatic β cell KATP channel, respectively) or abnormalities in the enzymes glucokinase, glutamate dehydrogenase and short chain acyl-CoA dehydrogenase ( SCHAD ). Loss of function mutations in the genes ABCC8 and KCNJ11 cause the most severe forms of HH which are usually medically unresponsive. HH is also observed in newborns with intrauterine growth retardation, in infants with perinatal asphyxia, in infants of diabetic (gestational and insulin dependent) mothers, in some infants with Beckwith-Weidemann syndrome and, more recently, in infants with no predisposing factors.3–6 In most of these conditions, the HH is a transient phenomenon and resolves spontaneously. However, some small for gestational age and appropriate for gestational age infants can have prolonged hyperinsulinaemic hypoglycaemia which requires treatment with diazoxide, persists for several months and then resolves spontaneously.7,8 Recognition of this group of patients is important as they are fully responsive to treatment with oral diazoxide. The underlying mechanisms of the HH in these patient groups are unclear at present. The biochemical basis of HH (congenital and secondary) involves dysregulated insulin secretion with …

Treating patients not numbers: the benefit and burden of lowering TSH newborn screening cut-offs

A century has passed since children affected by congenital hypothyroidism (CHT) were first treated successfully with thyroid extracts. At that time the diagnosis of severe ‘sporadic’ cretinism was based solely on clinical observation, long before measurement of thyroid hormone and thyroid stimulating hormone (TSH) was possible. Treatment success was evident with the dramatic clinical improvement in the growth and activity of these children.1 However, the severe mental retardation failed to improve in those early cases, which led to the conclusion that thyroid hormone is critical for normal neuronal maturation within a critical time window early in life. The introduction of newborn screening for CHT – by measuring TSH or T42 – finally offered a normal mental outcome to children who otherwise would be severely mentally disabled. Actual studies in adult patients from those first screening programs show that early treatment results in a normal IQ in more than 90% of affected patients.3 4 The treatment of CHT based on newborn screening is therefore one of the most successful achievements in paediatrics. As always, such a success bears the risk of ‘overdoing it’, as the current discussion on TSH cut-offs points out. Stimulated by that early success and with the intention of further improving the programs, the TSH cut-off levels were lowered to facilitate the diagnosis of milder, previously ‘missed’ cases of CHT. Starting with 50 mU/l TSH as the cut-off in the early programs, which were able to identify severe cases of CHT, the current debate discusses a cut-off value as low as 5 mU/l. A new publication on this critical cut-off range was recently published in the Archives of Disease in Childhood by Korada et al from Newcastle, UK5. Applying …

[18F]-DOPA Positron Emission Tomography for Preoperative Localization in Congenital Hyperinsulinism

In recent years, considerable progress has been made in the biochemical, morphological and molecular genetic differentiation of congenital hyperinsulinism (CHI). Fluorine-18 L-3,4-dihydroxyphenylalanine positron emission tomography (18F-DOPA-PET) has been introduced for differentiation between focal and diffuse CHI. The ability to take up L-DOPA and convert it into dopamine is correlated with the activity of the aromatic amino acid decarboxylase and increased in the hyperfunctional affected pancreatic area in comparison to normally functioning pancreas. The high sensitivity of this method allows the surgeon to perform a curative limited resection of a focus without the risk of long-term diabetes. The exact preoperative planning by 18F-DOPA-PET/CT computer tomography allows laparoscopic operation in selected cases with the focus in the tail and limits necessity to open the pancreatic duct in cases with focus in the head. Patients with persistent CHI should be managed within a strong network of diagnostic, treatment, and research institutions.

Long-Term Non-Surgical Therapy of Severe Persistent Congenital Hyperinsulinism with Glucagon

Background: Congenital hyperinsulinism (CHI) is characterized by severe hypoglycemia caused by dysregulated insulin secretion. The long-term outcome is dependent on prevention of hypoglycemic episodes to avoid the high risk of permanent brain damage. Severe cases are usually resistant to diazoxide or nifedipine. In addition, somatostatin analogues are ineffective in a subgroup of patients to achieve stable euglycemia. In these infants the only remaining long-term option has been subtotal pancreatectomy with high risk of diabetes mellitus. Intravenous infusions of glucagon are used as immediate treatment to stabilize euglycemia in affected newborns. The rationale of this treatment comes from the observation of an increased glycogen content of the liver when glycogenolysis is inhibited by insulin. Objective: To review the efficacy and safety of long-term subcutaneous glucagon infusion as a potential therapeutic option for blood glucose stabilization in infants with severe CHI without the need of additional intravenous glucose or immediate surgical intervention. Method: Retrospective review of 9 children with CHI who received continuous subcutaneous infusion of glucagon for weeks or months. Glucagon was added to octreotide to replace octreotide-induced suppression of endogenous glucagon secretion, thereby liberating glucose by stimulation of hepatic glycogenolysis. In 3 cases, a stabilized formulation of glucagon was used to prevent glucagon crystallization that frequently occurs in smaller volumes. Results: Introduction of glucagon allowed the reduction or discontinuation of central glucose infusion in all children studied. In 2 patients, glucagon was introduced due to recurrent hypoglycemia despite subtotal pancreatectomy. Six out of 9 children were discharged home on this treatment, which their parents were able to continue without further symptomatic hypoglycemia, convulsions or unconsciousness. In 3 children, subcutaneous glucagon was continuously administered for 1–4 years leading to stable euglycemia. However, 2 children with diffuse type still required subtotal pancreatectomy. As a possible side effect, 2 children developed erythema necrolyticum, which resolved after discontinuation of the glucagon infusion. This has been described before in glucagonoma. Conclusion: In this retrospective series, combination therapy of low-dose octreotide and subcutaneous glucagon infusion has been effective in preventing hypoglycemic episodes in severe CHI. We propose this may serve as a therapeutic option in place of high rates of glucose infusion through a central venous catheter and as an alternative to subtotal pancreatectomy in diffuse type of CHI.

A New C-Terminal Located Mutation (V272ter) in the PIT-1 Gene Manifesting with Severe Congenital Hypothyroidism

Objective: We describe a newborn with clinical signs of severe hypothyroidism and combined pituitary hormone deficiency due to a new mutation in the PIT-1 gene. Patient and Methods: Endocrine stimulation test revealed a deficiency for PRL, TSH and GH, suggesting a defect in the pituitary transcription factor PIT-1. Genetic analysis of the PIT-1 gene was performed by exon-specific PCR, followed by SSCP mutation screening and DNA sequencing of the abnormal migrating fragments. Results: DNA sequencing revealed a new mutation (V272ter) in direct neighborhood to a known mutational hot spot (R271W) in the C-terminal part of the PIT-1 molecule. Conclusions: Whereas the R271W mutation has a dominant negative effect on the mutant protein, the newly described mutation is inherited in an autosomal-recessive way. The biological consequences of these two different mutations are discussed.

Does the aromatic l-amino acid decarboxylase contribute to thyronamine biosynthesis?

Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase.

Harmonization of growth hormone measurements with different immunoassays by data adjustment.

Abstract Background: The aim of our study was to evaluate the between-assay variability of commercially available immunoassays for the measurement of human growth hormone (hGH). In addition, we asked whether the comparability of the diagnosis of childhood onset growth hormone deficiency could be improved by adjusting hGH results by statistical methods, such as linear regression, conversion factors, and quantile transformation. Methods: In archived sera from 312 children and adolescents (age: 17 days–17 years) hGH values between 0.01 and 16.5 ng/mL were determined by using the following immunoassays: AutoDELFIA (PerkinElmer), BC-IRMA (Beckman-Coulter), ELISA (Mediagnost), IMMULITE 2000 (Siemens), iSYS (IDS), Liaison (DiaSorin), UniCel DxI 800 Access (BeckmanCoulter) and “In house”-RIA (Tübingen). Results: The assays differed in median hGH concentrations by as much as 5.44 ng/mL (Immulite), and as little as 2.67 ng/mL (BC-IRMA). The mean difference between assays ranged from 0.35 to 2.71 ng/mL, whereas several samples displayed differences up to 11.4 ng/mL. The best correlation (r=0.992) was found between AutoDELFIA and Liasion, the lowest (r=0.864) was between an in-house RIA and iSYS. The between-assay CV (mean±SD) of values within the cut-off range was 24.3%±7.4%, resulting in an assay-dependent diagnosis of growth hormone deficiency (GHD) in more than 27% of patients. Yet, adjustment of this data by linear regression or a conversion factor reduced the CV below 14%, and the ratio of assay-dependent diagnoses below 8%. Using quantile transformation, the CV and ratio were reduced to 11.4% and <1%, respectively. Conclusions: hGH measurements using different assays vary significantly. Linear regression, conversion factors, or particularly quantile transformation are useful tools to improve comparability in the diagnostic procedure for the confirmation of GHD in childhood and adolescence.