Investigation inborn errors of Metabolism Flashcards
What are strategies emplyed for investigations of IEMs?
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
How are symptomatic patients investigations?
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
What is the common clinical presentation of IEM?
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
What are some routes of testing for IEMs?
- Metabolite test
- More complicated metabolite testing
- Enzyme analysis/Functional studies
- Mutation/Gene analysis
Why are Genetics not used as the 1st line of testing?
- 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
What is the traditional route of testing for IEMs?
- 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
What are the types metabolites tested for in IEMs?
- 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
What are techniques used in the analysis of IEMs?
- Chromatography
- Mass-spectrometry
- Electrophoresis
- Enzyme assays
What is Chromatography?
- Compounds separated due to their differing interactions between a mobile and stationary phase:
What are examples of Chromatography?
- Thin layer chromatography (TLC) – 1D and 2
- HPLC
- UPLC
- Ion exchange
What are features of Thin Layer technique?
Simple technique
- Qualitative
- Gives (relatively) quick screen but
- Subjective
- May miss more subtle patterns
What are Principles of Thin layer chromatography (TLC)?
- 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)
What is 1-Directional Thin Layer Chromatography for Oligosacharides?
- 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
Describe a 2D urine TLC?
- Plate run in different solvents in different directions
- Separates amino acids that would co-chromatograph if only run in a single direction
What are examples of Mass Spectrometry?
- Mass-spectrometry
- LC-MS/MS
- GC-MS
What are features of Mass Spectrometry?
- 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)
Describe the use of an enzyme assay?
- 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)
What are issues with measuring enzyme assays?
Sample collection often more difficult
- Enzymes often labile
- Sample is invasive
What are examples of enzyme assays?
- Biotinidase Deficiency – assess plasma biotinidase activity
- Lysosomal enzymes
What are features of Biotinidase deficiency?
- 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
How are Lysosomal Enzyme Assays undertaken?
- 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)
What are problems with Biotinidase deficiency assays?
- 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