Biochem. gene. & NBS

Question 1

Why is it easier to screen for inborn errors of metabolism using techniques such as liquid chromatography and mass spectrometry than by analysis of genetic material?

Answer 1

• quicker to get results than genetic analysis
  bc less complicated than looking for abnormalities in genetics e.g. mutations, pseudogenees, SNPs
• in biochem easy to find abnormal effects from abnormal genes

Question 2

What is the difference between inborn errors of metabolism and newborn screening?

Answer 2

Inborn errors: disturbances in metabolic pathways from genetic defects
NBS: pre-symptomatic detection & treatment of metabolic genetic disease before matabolic decompensation

Question 3

Describe what is involved in analysis of samples by mass spectrometry

Answer 3

1. Fragmentation of molecule
2. Ionisation of fragments
3. Selection of ion (m/z)
4. Quantification of ion

Question 4

What sample collection technique and analytical assays are used in newborn screening programs for the following: PKU, congenital hypothyroidism, cystic fibrosis, FA disorders, galactosemia

Answer 4

Collections: Heel prick blood spot on cellulose (guthrie) card & dried
Assays for:
- PKU: bact. inhibit. agar –> MSMS
- C.Hypothyroid.: Immunoassay
- CF: Immunoassay & DNA
- FA disorders: TMS/MS
- galactosemia: Enz assay

Question 5

What is PKU, what causes this condition, and what toxic metabolites are responsible for the symptoms.

Answer 5

Phenylketonuria (PKU)
- Autosomal recessive mutation in phenylalanine hydroxylase enz:
- Phe -x-> Tyr
=> build up of Phenylketones (phe-pyruvic acid & -lactic acid) in blood & urine

Question 6

Are inborn errors of metabolism due to single gene defects or are they polygenic, i.e. due to the influence of multiple genes +/- environmental influences?

Answer 6

No, inborn errors are monogenic (single gene defects)

Question 7

What techniques are common in both inborn errors of metabolism and NBS?

Answer 7

• LCMSMS
• MS

Question 8

how does LC-MS differ from MS-MS (see slides 38-42), at ~43-55 mins on the Inborn Errors of Metabolism lecture.

Answer 8

LCMS: LC separation of compounds-> ID parent & daughter ions by LC retention time-> quantification against Std
MSMS: Ionisation (e- spray ionis.)-> parent molecules sorted & selected in 1st MS -> fragmentation -> sorted & selected in 2nd MS
*using HPLC in MSMS (LCMSMS) improves Sp

Question 9

levels of plasma phenylalanine for normal & PKU

Answer 9

N: <137 umol/L
PKU: 1000-2000 umol/L at 5 days old

Question 10

biochemical markers for disease & from type of specimen

Answer 10

• AA from urine, plasma, CSF
• organic marker from urine
• Carnitine & acylcarnitine from plasma & urine
• Metabolic disease targets from plasma, urine

Question 11

What is MCAD & detection method & biochemical feature

Answer 11

• common FA oxidation defect
• MSMS
• Hypoketotic hypoglycemia