Investigation inborn errors of Metabolism

Question 1

What are strategies emplyed for investigations of IEMs?

Answer 1

Pre-symptomatic Screening

• can be whole population (e.g. newborn screening) or selected “at risk” groups

Investigation of symptomatic patient

• Presentation with set of clinical symptoms
• Clinical picture directs testing

Question 2

How are symptomatic patients investigations?

Answer 2

Clinical picture directs testing

• Symptoms (current)
• Symptoms (previous history)
• Family history (family members with similar history?, parents related?)
• Imaging / non-biochemistry tests

“Routine” biochemistry tests can provide key information:

• To acute management of some scenarios (e.g. ammonia, glucose,)
• To which tests should be performed next

Question 3

What is the common clinical presentation of IEM?

Answer 3

Acute:

• Hypoglycaemia
• Hyperammonaemia
• Metabolic Acidosis
• Fits and seizures
• Rhabdomyolysis
• Liver failure
• Encephalopathy

Chronic:

• Developmental delay
• Intellectual disability
• Learning difficulties
• Chronic organ problems – cardiomyopathy, skeletal problems, muscle disorders

Question 4

What are some routes of testing for IEMs?

Answer 4

• Metabolite test
• More complicated metabolite testing
• Enzyme analysis/Functional studies
• Mutation/Gene analysis

Question 5

Why are Genetics not used as the 1st line of testing?

Answer 5

• Cost/time (but both rapidly reducing with next generation sequencing (NGS))
• Can’t always ompletely exclude disorders with genetics alone as not all mutations maybe covered (+large deletions etc)
• Significance of mutation not always known, often poor genotype phenotype relationship

Question 6

What is the traditional route of testing for IEMs?

Answer 6

• Clinical presentation/basic lab tests
• Genetics: WES*/Target gene panels/?100k genome project
• Pathogenic variant in relevant
• VOUS in one or more genes. Diagnostic metabolic tests

Question 7

What are the types metabolites tested for in IEMs?

Answer 7

• Amino acids (amino acid disorders)
• Organic acids (organic acidurias)
• Acylcarnitines (fat oxidation defects)
• Mucopolysaccharides/Oligisaccharides (some lysosomal storage disorders)
• Very long chain fatty acids (VLCFA) (peroxisomal disorders)
• Gal-1-PUT (screening test for classical galactosaemia

Question 8

What are techniques used in the analysis of IEMs?

Answer 8

• Chromatography
• Mass-spectrometry
• Electrophoresis
• Enzyme assays

Question 9

What is Chromatography?

Answer 9

• Compounds separated due to their differing interactions between a mobile and stationary phase:

Question 10

What are examples of Chromatography?

Answer 10

• Thin layer chromatography (TLC) – 1D and 2
• HPLC
• UPLC
• Ion exchange

Question 11

What are features of Thin Layer technique?

Answer 11

Simple technique

• Qualitative
• Gives (relatively) quick screen but
• Subjective
• May miss more subtle patterns

Question 12

What are Principles of Thin layer chromatography (TLC)?

Answer 12

• Sample seeded onto a plate with coated with a stationary phase (e.g. silica)
• Plate placed in a closed tank containing solvent mixture
• As solvent runs up the plate compounds are separated dependent on interaction with stationary phase
• Can run plate in 1 or 2 directions (1D or 2D)

Question 13

What is 1-Directional Thin Layer Chromatography for Oligosacharides?

Answer 13

• Urine seeded onto silica gel coated glass plates
• Plate run in solvent in one direction
• Stained with orcinol
• Pattern of oligosaccharides can be indicative of certain lysosomal storage disorders

Question 14

Describe a 2D urine TLC?

Answer 14

• Plate run in different solvents in different directions
• Separates amino acids that would co-chromatograph if only run in a single direction

Question 15

What are examples of Mass Spectrometry?

Answer 15

• Mass-spectrometry
• LC-MS/MS
• GC-MS

Question 16

What are features of Mass Spectrometry?

Answer 16

• Coupling with chromatography leads to a high degree of specificity
• Can give you qualitative (e.g. organic acids, acylcarnitines) and/or quantitative (e.g. purines, amino acids, acylcarnitines, creatine, etc etc)

Question 17

Describe the use of an enzyme assay?

Answer 17

• Direct measurement of (potentially) affected enzyme in metabolic pathway
• Sample type must reflect where enzyme is expressed (e.g. leucocytes (lysosomal enzymes), muscle (respiratory chain enzymes))
• Need a labelled substrate which can be measured (fluorimetrically, spectrophotometry, radio-labelled, MS/MS)
• Reaction condition very important (temperature, pH etc)

Question 18

What are issues with measuring enzyme assays?

Answer 18

Sample collection often more difficult

• Enzymes often labile
• Sample is invasive

Question 19

What are examples of enzyme assays?

Answer 19

• Biotinidase Deficiency – assess plasma biotinidase activity
• Lysosomal enzymes

Question 20

What are features of Biotinidase deficiency?

Answer 20

• Simple colorimetric assay
• Biotinidase in sample cleaves the amide bond of the substrate, biotin-p-amidobenzoic acid (PABA)
• This releases biotin and PABA
• PABA converted to a purple azo dye which can be quantitated by measuring its absorbance at 546nm

Question 21

How are Lysosomal Enzyme Assays undertaken?

Answer 21

• Most done in leucocytes
• Leucocyte preparation time consuming and technically difficult (and must be done within 24-48 hours of sampling)
• Measure a reference enzyme to check for sample viability
• Can measure single enzyme or panels (White Cell Enzyme screen)

Question 22

What are problems with Biotinidase deficiency assays?

Answer 22

• Enzyme is labile (reduction of activity of approx 35% per day at RT)
• Not very sensitive – but often good enough, interpretet in light of clinical picture and other tests (organic acids abnormal