Hypoglycemia: Clinical and molecular aspects

1. Hypoglycemia: Clinical and molecular aspects J Lebl and collaboratorsDepartment of Pediatrics, 2nd Faculty of MedicineCharles University in Prague, Czech Republic

2. Glucose • Central role in fuel economy • Provides 38 mol of ATP / mol of glucose oxidised • Key substrate for energy metabolism of brain cells. • Cerebral glucose uptake • Facilitated diffusion dependent on actual BG level.

3. Hypoglycaemia  Hypoglycorhachia (low glucose in CSF)  Energy deficit of brain cells  EMERGENCY

4. Blood glucose regulation FATTY ACIDS AMINOACIDS MEALS GLUCONEOGENESIS and alternative energy sources (80 %: beta-oxidation) BLOOD GLUCOSE GLYCOGEN SYNTHESIS GLYCOGENOLYSIS LIVER GLYCOGEN stores for 6-12 hours of fasting BRAIN

5. Blood glucose regulation FATTY ACIDS AMINOACIDS MEALS GLUCONEOGENESIS and alternative energy sources (80 %: beta-oxidation) Insulin - GLYCOGEN SYNTHESIS Insulin + (Cortisol, GH - ) GSD 0 - BLOOD GLUCOSE GLYCOGENOLYSIS Insulin - Glucagon, epinephrin + (Cortisol, GH + ) GSD I, III, VI, IX - LIVER GLYCOGEN stores for 6-12 hours of fasting BRAIN

6. Sophia

7. Sophia • First baby of healthy parents • Normal pregnancy, born at 39th gestational week, 4000g, 52cm (large for gestational age); AS 9-10-10 • At age 38 hours sudden hypotonia, subsidence. BG 1.2 mmol/L (22 mg/dL) • Recurrent hypoglycemias despite of IV glucose (up to 13.5 mg/kg/min) Patient‘s photos were removed from this version

8. = Congenital hyperinsulinism • Transferred to neonatal ICU: BG 0.9 mmol/L (16 mg/dL), insulin 103.2 mIU/L • Catheter sepsis, intensive antibiotic therapy • Referred for endocrine evaluation at age 11 days • At admission, BG 1.1 mmol/L (20 mg/dL): “Critical blood sample”: • beta-hydroxybutyrate 0.1 mmol/L • insulin 13.7 mIU/L; C-peptide 1604 pmol/L • cortisol 482 nmol/L • growth hormone 14.20 ug/L • triglycerides 1.78 mmol/L; lactate 3.74 mmol/L

9. Pharmacological treatment • Diazoxid (Proglicem) 12.5 mg/kg/day since day 1, in four daily doses. IV glucose was stopped, however hypoglycemias recurred after several days • Octreotid (Sandostatin) 20 ug/kg/day added at day 6, in four daily doses SC. Milk formula was fortified by maltodextrin. BG levels remained close to normal range – Sophia was discharged from hospital • Three months later relapse of frequent hypoglycemias – dose of octreotide increased to25 ug/kg/day.

10. feeding every 3 hrs • BG measurement every 3 hrs • SC and oral medication every 6 hrs • Neuropsychological development normal • EEG normal • weight gain normal Patient‘s photos were removed from this version

11. Genetic testing • Sophia carries a heterozygous mutation of ABCC8 gene (IVS10-10T>G)that encodes SUR1 subunit of beta-cell KATP channel • No mutations were found in KCNJ11, GCK, HNF4A • ABCC8 IVS10-10T>G is a novel intronic variant located prior to exon 11 that is essential for gene splicing and is evolutionary highly conserved. The T>G substitution probably leads to skipping of exon 11.

12. Postzygotic (somatic) uniparental isodisomy of paternally inherited mutation.Results in focal pancreatic lesion with hyperinsulinism.

13. 18F-DOPA PET/CT

14. Surgery At age 5 months, Sophia underwent partial pancreatic resection (of cauda and part of pancreatic body)

15. Histology Hyperplastic and irregular pancreatic islets, partially confluent

16. Sophia at age 8 months • Normal BG (3.9 – 5.9 mmol/L; 70-106 mg/dL) • HbA1c 32 mmol/mol • Stool elastase 411 ug/g • Normal development • EEG normal • No medications • Feeding 6times daily with night pause Patient‘s photos were removed from this version

17. Congenital hyperinsulinism - PHHI Persistent Hyperinsuliemic Hypoglycemia of Infants Glucose Kir6.2 SUR1 GLUD1 HADH GCK HNF4A HNF1A SLC16A1 GLUT2 5 GCK SUR1 6 7 Kir6.2 2 G-6-P 1 3 K+ GLUD1 - HADH ATP ADP 4 + Ca2+ Insulin Depolarisation 8 SLC16A1 Ca2+

18. Congenital hyperinsulinism (PHHI) Channelopathies • ABCC8- encoding SUR1 Metabolopathies • GLUD1 - encoding mitochondrial enzyme glutamate dehydrogenase (GDH); PHHI with hyperammonemia • HADH - encoding mitochondrial enzyme short-chain L-3-hydroxyacyl-CoA dehydrogenase; interacts with GDH • GCK - encoding glucokinase (activating mutation, AD) Transcription factors • HNF4A – inactivating mutation (AD) – later in life: MODY1 • HNF1A– inactivating mutation (AD) – later in life: MODY3 Transporters • SLC16A1 – carries lactate and pyruvate across cell membrane • KCNJ11 - encoding Kir6.2

19. Sites of action for Diazoxide, Nifedipine and Octreotide Glucose GLUT2 somatostatin receptor GCK SUR1 G-6-P Kir6.2 K+ - ATP ADP + Ca2+ Insulin Depolarisation Ca2+ Diazoxide Nifedipine Octreotide Khalid Hussain, Meet the Expert, ESPE 2013

20. Management cascade • Medical therapy • Diazoxide (up to 20mg/kg/day) • Nifedipine (2.5mg/kg/day) • Glucagon • Octreotide (somatostatin analogue) Khalid Hussain, Meet the Expert, ESPE 2013

21. Diazoxide Responsiveness: Definition • Normal fasting tolerance • Normal feeding volume and frequency • No hypoglycemia! • Appropriate increase in fatty acids and ketone bodies during fasting test Khalid Hussain, Meet the Expert, ESPE 2013

22. Focal form of CHI Paternally transmitted recessive SUR1 or Kir6.2 mutation plus somatic loss of maternal allele within the focal pancreatic lesion. Diagnosis: 1. DNA - typical genotype 2. Topical diagnosis – PET (using 18F-fluoro-L-DOPA) Therapy: Segmental resection of focal lesion (laparoscopic)

23. Elisabeth

24. Elisabeth was born to healthy unrelated parents. Her brother is two years older. • Elisabeth was growing much just before birth. Foetalultrasound showed: • macrosomia • hepatomegaly • nephromegaly. Patient‘s photos were removed from this version

25. At 34 week gestation, Caesarean section was done due to macrosomia: • 3790 g / 54 cm • Apgar score 6-3-7, dyspnoeic due to significant macroglossia. Her status improved following oxygen administration Patient‘s photos were removed from this version

26. First day of life: Asymptomatic hypoglycaemias 1.3 mmol/L (23 mg/dL) • Insulin 20.3 mIU/L in the same blood sample •  HYPERINSULINAEMIC HYPOGLYCAEMIA • IV glucose infusion given initially; later on, blood glucose levels stabilised if maltodextrin was added to food. • After 3 weeks, BG was declining again, up to 1.7 mmol/L (31 mg/dL). Elisabeth was transferred to our centre. • We started administration of diazoxide (Proglicem) and subsequently of octreotide (Sandostatin).

27. Ear abnormalities • Macroglossia • Umbilical abnormalities • Visceromegaly • Hyperinsulinism • Large for gestational age • Large placenta • Long umbilicus • Polyhydramnion Patient‘s photos were removed from this version Beckwith-Wiedemann syndrome(exomphalos-macroglossia-gigantism, EMG)

28. Diagnosis

29. Patient‘s photos were removed from this version BWS: Etiology • Chromosomal imprinting of short arm of 11th chromosome, region 11p15.5: • trisomy with paternal duplication • paternal disomy with loss of maternal chromosome. • Results in overexpression of INS gene (encoding for insulin) and of IGF2 gene (encoding for IGF-II – a growth promotor). • Expression of some tumour-supressor genes may be compromised.

30. High risk of solid tumours within the first years of life: • nephroblastoma • adrenal carcinoma • hepatoblastoma • rhabdomyosarcoma • neuroblastoma Patient‘s photos were removed from this version

31. Elisabeth: alpha-fetoprotein level Age (months)

32. Patient‘s photos were removed from this version • The combined therapy allowed to stabilise BG levels and further on to stop Diazoxide administration. • However, significant macroglossia compromised swallowing and lead to hypoxaemia. Finally, it was necessary to reduce tongue size surgically and to re-educate swallowing afterwards.

33. Patient‘s photos were removed from this version BWS: Outcome • Mortality 20-25 % within the first year • Heart defects • Hypoglycaemia • Tumours • Intellectual development depends predominantly on the damage caused by periods of hypoglycaemia • Long-term growth mostly over the 95. percentile • Following the infant period, hypoglycaemias resolve and the outcome improves • As to expected incidence 1:10000-1:15000, most cases remain undiagnosed.

34. Age 17 months - CT

35. Margaret

36. Margaret • Normal pregnancy, born at 40th week, 3020g/50 cm. Apgar score 9-10-10 • 2nd day of life - tonic seizures, apnoea • BG 1.2 mmol/L (22 mg/dl) • Following a short-term glucose infusion, symptoms resolved. No further investigation was done

37. At 8 months, seizures of all extremities and a transitory unconsciousness in the morning • she improved after tea with sugar • no lab tests • investigated by a neurologist • EEG: slightly abnormal findings • Dx Epilepsy – antiepileptic medication started • Recurrent seizures, mostly during intercurrent illnesses • Dx Epilepsy resistant to therapy

38. At 7.5 years, height 106.5 cm (-3.4 SD) • Referred for evaluation of short stature

40. At 7.5 years, height 106.5 cm (-3.4 SD) • Referred for evaluation of short stature • IQ 74 • spontaneous hypoglycemia 1.8 mmol/L (32 mg/dL) following twice vomiting • at hypoglycemia (critical sample) • insulin 0.2 mIU/L • C-peptide 35 pmol/L • GH < 1.5 mIU/L • cortisol 180 nmol/L • Dx: Growth hormone deficiency, TSH deficiency, ACTH deficiency – combined pituitary hormone deficiency

41. Impaired vision of the right eye • VOP 6/6-9 • VOL 6/60-36 • Fundoscopy: pale optic nerve papils

42. MRI

43. Septo-optic dysplasia • optic hypoplasia, visual impairment • mid-line defects • pituitary hormone deficiency

44. Growth hormone L-thyroxine Hydrocortisone Estradiol Estradiol + progestin

45. Congenital hypopituitarism Hormonal deficiency GH  IGF-I ACTH  cortisol TSH  thyroxine FSH, LH (ADH) Neonatal symptoms hypoglycemia prolonged jaundice testicular retention (dehydration) }

46. Ann

47. Ann Patient‘s photos were removed from this version • Ann is 17 years old. She was admitted to hospital with unconsciousness and convulsions last year before Christmas. Severe hypoglycemia 1.1 mmol/L (20 mg/dL) was measured at admission. She quickly recovered following glucose infusion. Medical history • She was loosing weight and was investigated for anorexia nervosa in spring and summer. • The night before admission, she celebrated graduation with her schoolmates and drank some alcohol (not much).

48. Patient‘s photos were removed from this version Ann Medical history cont‘ed • Ann had been jumping on a trampoline competitively. Half year ago, she stopped sports because she lost the strength and felt tired • During summer, she was working in a supermarket, had to get up early and to work hardly. She felt dizzy, could not eat and vomited repeatedly. According to descriptions of anorectic symptoms in journals for young girls, she believed to suffer from anorexia nervosa. She lost 7 kg weight. • One day she collapsed. At hospital, she had blood glucose 1.9 mmol/L (34 mg/dL) but was discharged home after glucose infusion.

49. Patient‘s photos were removed from this version Ann Medical history cont‘ed • For severe hypoglycemia, she was referred for examination at an endocrinologist for adults • He classified her as a clear case of anorexia nervosa and referred to a psychiatrician • Finally, she had prescribed antidepressant medication but was not taking the pills

50. Patient‘s photos were removed from this version Ann Ann noticed that she caught a sun in April at her first visit to a swimming pool. Since then, she kept a new, pretty nice skin color. She was happy about it but noticed an unusual distribution of skin pigment around her nails