CSF Testing in Neurotransmitter Disorders

1. CSF Testing in Neurotransmitter Disorders Keith Hyland, Ph.D. Tel, 678-597-5659 khyland@medicalneurogenetics.com khyland@mnglab.com

2. Conflict of Interest Disclosure

3. Topics to be Covered Pathways Methods Neurotransmitter Disorders Other Disorders Detected.

4. When to Perform a Lumbar Puncture Standard screening tests are likely normal Encephalopathy of unknown etiology Seizures of unknown etiology �Cerebral Palsy� of unknown etiology Unexplained movement disorder Explain that not all of these will have seizures but LP is required for diagnosisExplain that not all of these will have seizures but LP is required for diagnosis

5. Disorders of Neurotransmitter Metabolism Catecholamine (dopamine, and norepinephrine) Serotonin

6. Sample Collection Sample collection is critical! Rostro - caudal gradients Stability of metabolites (particularly tetrahydrobiopterin) Always call the lab for instructions (678- 597- 5658)

9. HPLC DIAGNOSIS OF GTP-CYCLOHYDROLASE DEFICIENCY

10. GTP-Cyclohydrolase Deficiency Problems with Diagnosis? No hyperphenylalaninemia Same CSF metabolite pattern can be seen following ischemia / hypoxia Same CSF pattern can be seen following insult to dopaminergic cells

11. Definitive Diagnosis Fibroblast enzyme assay Genomic sequencing

13. HPLC Diagnosis of Sepiapterin Reductase Deficiency

14. Caveats? Dihydropteridine reductase deficiency can lead to similar metabolite changes. Definitive diagnosis: Fibroblast enzyme assay Genomic sequencing Sepiapterin in CSF

16. HPLC DIAGNOSIS OF TYROSINE HYDROXYLASE DEFICIENCY

19. HPLC Diagnosis of AADC Deficiency

20. Aromatic L-Amino Acid Decarboxylase Deficiency � Definitive Diagnosis? Plasma enzyme assay Mutation analysis available

22. Pyridox(am)ine 5'-phosphate Oxidase Deficiency.PNPO � Gene map locus 17q31.22 CSF threonine, +/- glycine CSF 3-OMD CSF 5HIAA, CSF HVA Urinary vanillactic acid

23. Pyridox(am)ine 5'-phosphate Oxidase Deficiency. L-dopa Dopamine AADC AADC requires vitamin B6 as a cofactor Treatment with pyridoxine NO EFFECT

26. Pyridoxal 5�-phosphate Responsive Seizures Treatment with pyridoxal 5� phosphate (10 � 50 mg/kg/day) Immediate cessation of seizures

29. Pyridoxal 5�-phosphate Responsive Seizures Defect lies in the conversion of pyridoxine to pyridoxal 5�-phosphate Pyridoxamine Phosphate Oxidase (PNPO) Deficiency Clayton PT et al Lancet 2003

30. Pyridoxal 5�-phosphate Responsive Seizures CSF pyridoxal 5�-phosphate concentrations are low. Ormazabal et al 2008

31. Folinic Acid Responsive Seizures Serendipity and Science? Explain the way that the seizure disorder was first discoveredExplain the way that the seizure disorder was first discovered

32. CSF Neurotransmitter Metabolite(HPLC � electrochemical detection)

33. Etiology

34. Serendipity and Science Explain the way that the seizure disorder was first discoveredExplain the way that the seizure disorder was first discovered

35. Pyridoxine responsive seizures Mutations in antiquitin in individuals with pyridoxine-dependent seizures. Mills et al Nat Med 2006

36. Pathophysiologic mechanism in pyridoxine responsive seizures

37. Relationship between AASA and unknown

38. Pyridoxal 5�-phosphate Responsive Seizures Pyridoxal 5'-phosphate may be curative in early-onset epileptic encephalopathy. Hoffmann et. al. J. Inherit Metab Dis 2007

39. Test To Request

40. Conclusions All neonates and infants with intractable seizures of unknown origin should have a lumbar puncture performed and neurotransmitter metabolites analyzed. Pyridoxal 5�-phosphate responsive seizures Folinic acid responsive seizures/Pyridoxine responsive seizures

41. The vesicular amine transporter acts via an ATP-dependent process linked to a proton pump. VAT has a dual role :Maintain a ready supply of neurotransmitter and Mediate release. Action potential arrives, CA2+ channels open. Get calcium influx. The increased calcium promotes fusion of the vesicle with the neuronal membrane. DA reuptake by the DAT is energy dependent coupled to a NA+ gradient across the neuronal membrane Action is via G protein- coupled receptors. These are many types and en masse are termed the G protein receptor-linked superfamily. The slide depicts dopamine binding to two different receptors leading to either the inhibition (D2,3,4) or stimulation (D1, D5) of adenylate cyclase. Mechanism in both cases involves GTP binding to the G protein, change in activity of adenylate cyclase and a change in intracellular cyclic AMP levels which in turn regulate the activity of protein kinase A. G protein coupled receptors can also act by altering phosphoinositide hydrolysisleading to protein kinase C activation and release of CA2+. HOW DO RELEASE MODULATING AUTORECEPTORS WORK? The vesicular amine transporter acts via an ATP-dependent process linked to a proton pump. VAT has a dual role :Maintain a ready supply of neurotransmitter and Mediate release. Action potential arrives, CA2+ channels open. Get calcium influx. The increased calcium promotes fusion of the vesicle with the neuronal membrane. DA reuptake by the DAT is energy dependent coupled to a NA+ gradient across the neuronal membrane Action is via G protein- coupled receptors. These are many types and en masse are termed the G protein receptor-linked superfamily. The slide depicts dopamine binding to two different receptors leading to either the inhibition (D2,3,4) or stimulation (D1, D5) of adenylate cyclase. Mechanism in both cases involves GTP binding to the G protein, change in activity of adenylate cyclase and a change in intracellular cyclic AMP levels which in turn regulate the activity of protein kinase A. G protein coupled receptors can also act by altering phosphoinositide hydrolysisleading to protein kinase C activation and release of CA2+. HOW DO RELEASE MODULATING AUTORECEPTORS WORK?

42. Many CSF samples have unexplained low HVA and 5-HIAA concentrations? Defective VMAT2 ? VMAT2 knockout shows low levels of dopamine, serotonin and metabolites Genomic sequencing of VMAT2 is available. No mutations found to date. Only 3 samples examined

43. The vesicular amine transporter acts via an ATP-dependent process linked to a proton pump. VAT has a dual role :Maintain a ready supply of neurotransmitter and Mediate release. Action potential arrives, CA2+ channels open. Get calcium influx. The increased calcium promotes fusion of the vesicle with the neuronal membrane. DA reuptake by the DAT is energy dependent coupled to a NA+ gradient across the neuronal membrane Action is via G protein- coupled receptors. These are many types and en masse are termed the G protein receptor-linked superfamily. The slide depicts dopamine binding to two different receptors leading to either the inhibition (D2,3,4) or stimulation (D1, D5) of adenylate cyclase. Mechanism in both cases involves GTP binding to the G protein, change in activity of adenylate cyclase and a change in intracellular cyclic AMP levels which in turn regulate the activity of protein kinase A. G protein coupled receptors can also act by altering phosphoinositide hydrolysisleading to protein kinase C activation and release of CA2+. HOW DO RELEASE MODULATING AUTORECEPTORS WORK? The vesicular amine transporter acts via an ATP-dependent process linked to a proton pump. VAT has a dual role :Maintain a ready supply of neurotransmitter and Mediate release. Action potential arrives, CA2+ channels open. Get calcium influx. The increased calcium promotes fusion of the vesicle with the neuronal membrane. DA reuptake by the DAT is energy dependent coupled to a NA+ gradient across the neuronal membrane Action is via G protein- coupled receptors. These are many types and en masse are termed the G protein receptor-linked superfamily. The slide depicts dopamine binding to two different receptors leading to either the inhibition (D2,3,4) or stimulation (D1, D5) of adenylate cyclase. Mechanism in both cases involves GTP binding to the G protein, change in activity of adenylate cyclase and a change in intracellular cyclic AMP levels which in turn regulate the activity of protein kinase A. G protein coupled receptors can also act by altering phosphoinositide hydrolysisleading to protein kinase C activation and release of CA2+. HOW DO RELEASE MODULATING AUTORECEPTORS WORK?

44. Many CSF samples have isolated low HVA concentrations Defective dopamine transporter (DAT)? DAT knockout shows low levels of dopamine and HVA Genomic sequencing of DAT is available. No mutations found. 6 samples examined

45. Dopamine transporter (DAT1) Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia. CSF HVA LEVELS ARE ELEVATED!! Kurian et al J Clin Invest 2009

47. Dopamine Transporter (DAT1) Deficiency Early onset Parkinsonian symptoms Pyramidal tract features Dystonia Global Developmental delay Normal MRI HVA/5HIAA ratio 5 to 13.4 Normal < 2.5 5HIAA within normal range.

48. Future Directions Serotonin transporter (SERT) Elevated 5HIAA?? Receptors � Pre and Post Synaptic Serotonin Dopamine Norepinephrine Next generation sequencing