Anti-Müllerian hormone and letrozole levels in boys with constitutional delay of growth and puberty treated with letrozole or testosterone.

STUDY QUESTION: Does treatment of constitutional delay of growth and puberty (CDGP) in boys with aromatase inhibitor letrozole (Lz) or conventional low-dose testosterone (T) have differing effects on developing seminiferous epithelium? SUMMARY ANSWER: Anti-Müllerian hormone (AMH) declined similarly in both treatment groups, and the two Sertoli cell-derived markers (AMH and inhibin B (iB)) exhibited differing responses to changes in gonadotrophin milieu. WHAT IS KNOWN ALREADY: Boys with CDGP may benefit from puberty-inducing medication. Peroral Lz activates gonadotrophin secretion, whereas intramuscular low-dose T may transiently suppress gonadotrophins and iB. STUDY DESIGN, SIZE, DURATION: Sera of 28 boys with CDGP who participated in a randomised, controlled, open-label trial at four paediatric centres in Finland between August 2013 and January 2017 were analysed. The patients were randomly assigned to receive either Lz (2.5 mg/day) (n = 15) or T (1 mg/kg/month) (n = 13) for 6 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 28 patients were at least 14 years of age, showed first signs of puberty, wanted medical attention for CDGP and were evaluated at 0, 3, 6 and 12 months of visits. AMH levels were measured with an electrochemiluminescence immunoassay and Lz levels with liquid chromatography coupled with tandem mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: AMH levels decreased in both treatment groups during the 12-month follow-up (P < 0.0001). Between 0 and 3 months, the changes in gonadotrophin levels (increase in the Lz group, decrease in the T group) correlated strongly with the changes in levels of iB (FSH vs iB, r = 0.55, P = 0.002; LH vs iB, r = 0.72, P < 0.0001), but not with the changes in AMH (P = NS). At 12 months, AMH levels did not differ between the groups (P = NS). Serum Lz levels (range, 124-1262 nmol/L) were largely explained by the Lz dose per weight (at 3 months r = 0.62, P = 0.01; at 6 months r = 0.52, P = 0.05). Lz levels did not associate with changes in indices of hypothalamic-pituitary-gonadal axis activity or Sertoli cell markers (in all, P = NS). LIMITATIONS, REASONS FOR CAUTION: The original trial was not blinded for practical reasons and included a limited number of participants. WIDER IMPLICATIONS OF THE FINDINGS: In early puberty, treatment-induced gonadotrophin stimulus was unable to counteract the androgen-mediated decrease in AMH, while changes in iB levels were associated with changes in gonadotrophin levels. AMH decreased similarly in both groups during the treatment, reassuring safety of developing seminiferous epithelium in both treatment approaches. Since a fixed dose of Lz induced variable serum Lz levels with a desired puberty-promoting effect in all boys, more research is needed to aim at a minimal efficient dose per weight. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Academy of Finland, the Foundation for Pediatric Research, the Emil Aaltonen Foundation, Sigrid Juselius Foundation and Helsinki University Hospital Research Funds. The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: NCT01797718.

Characterisation of new KATP-channel mutations associated with congenital hyperinsulinism in the Finnish population.

AIMS/HYPOTHESIS: ATP-sensitive potassium (K(ATP)) channels are crucial for the regulation of insulin secretion from pancreatic beta cells and mutations in either the Kir6.2 or SUR1 subunit of this channel can cause congenital hyperinsulinism (CHI). The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population. METHODS: Wild type or mutant Kir6.2 and SUR1 subunits were coexpressed in Xenopus oocytes. The functional properties of the channels were examined by measuring currents in intact oocytes or giant inside-out membrane patches. Surface expression was measured by enzyme-linked immunosorbance assay, using HA-epitope-tagged subunits. RESULTS: Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of K(ATP) current activation observed in response to metabolic inhibition in intact oocytes. The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 micro mol/l to 120 micro mol/l). The fourth mutation (K67N mutation in Kir6.2) did not affect surface expression nor alter the properties of K(ATP) channels in excised patches, but resulted in a reduced K(ATP) current amplitude in intact cells on metabolic inhibition, through an unidentified mechanism. CONCLUSION/INTERPRETATION: The four CHI mutations disrupted K(ATP) channel activity by different mechanisms. Our results are discussed in relation to the CHI phenotype observed in patients with these mutations.

Acute insulin response tests for the differential diagnosis of congenital hyperinsulinism.

Mutations in genes encoding the two subunits of the beta-cell ATP-sensitive potassium channel (K(ATP)) channel (SUR1 and Kir6.2) are the major cause of congenital hyperinsulinism (CHI). In this study, the K(ATP) channel genes were screened in a population-based study that included all verified Finnish CHI patients (n = 43) in a 27-yr period. Seven different mutations were identified, which accounted for 60% of all cases. The functional consequences of the major missense mutations were studied in vivo by determining acute (1-3 min) plasma insulin and C-peptide responses to calcium (n = 18), glucose (n = 12), and tolbutamide (n = 11) in those CHI patients who were able to take part in these studies. C-peptide and insulin responses to calcium were significantly higher in the patients with SUR1-E1506K mutation, compared with patients without K(ATP) channel mutations. The patients with SUR1-V187D mutation showed a reduced response to tolbutamide but unexpectedly did not show any response to calcium stimulation. A compound heterozygous patient with Kir6.2-(-54)/K67N mutations responded to calcium but also to tolbutamide. In conclusion, our results show that a positive response in the calcium test is indicative of a K(ATP) channel mutation, but all mutations cannot be identified with this method. The insulin response to tolbutamide in patients with SUR1 mutations is impaired to different extents, depending on the genotype. The combination of calcium and tolbutamide tests is a useful tool for the detection of CHI patients with K(ATP) channel dysfunction. Our results, however, also demonstrate the complexity of these responses and the difficulties in their interpretation.

K(ATP) channels and insulin secretion disorders.

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.

Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1.

ATP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic beta cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant K(ATP) channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.

A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.