Molecular Diagnosis of Galactosemia

• Molecular diagnosis of Galactosemia requires 4 stages

Galactosemia

• Classic galactosemia is an autosomal recessive disorder due to a deficiency in galactose 1-phosphate uridyltransferase (GALT).
• Neonates present with vomiting and diarrhea within days of milk intake.
• Most patients develop jaundice and hepatic failure which can be lethal if untreated.
• Newborn screening for galactosemia is routine in all states in the USA and many other countries.
• The main treatment is elimination of dietary galactose.
• This treatment does not prevent secondary complications such as growth and mental retardation, dyspraxia, cataracts, ataxia, and ovarian failure.

Galactose Metabolism

• Galactose converts to Galactose 1-phosphate via galactokinase.
• Galactose 1-phosphate converts to UDP-glucose via Galactose 1-phosphate uridyl transferase.
• UDP-glucose converts to UDP-galactose.
• UDP-galactose converts to UDP-glucose via epimerase.
• UDP-glucose facilitates glycogen synthesis/break down.
• Accumulation of Galactose 1-phosphate can cause liver damage and jaundice.
• Glucose and Galactose are C4 epimers.

GALT Enzyme Activity Assay

• GALT enzyme activity in red blood cells (RBC's) is assayed by measuring the conversion of [14C]-galactose-1-phosphate ([14C]-Gal-1-P) and UDP-glucose (UDPG) to glucose-1-phosphate (Glu-1-P) and 14C-UDP-galactose.
• The reaction is performed at 37°C for 1 hour using erythrocyte hemolysate normalized to hemoglobin content.
• In a proband with classical galactosemia, high levels of Gal-1-P [25.1 mg/dL (normal 1.0 mg%)] are present in the neonatal period.
• Absent activity of erythrocyte GALT [normal 36.3 1.7 moles/gHbg/(Hr)] is also observed in affected proband.

PCR-ELISA Test

• PCR-ELISA combines PCR and ELISA techniques to detect nucleic acid instead of protein.
• This assay quantifies the PCR product directly after immobilization with biotinylated DNA on a microplate.
• The principle is based on the interaction between DIG-labeled DNA sequencing and anti-DIG antibody.
• Probes that do not connect to the target DNA, if target DNA is incorrectly copied, will result in a negative answer.
• A specific gene part is selected, and primers are designed for it.
• Amplification is carried out using digoxigenin-11-dUTP (DIG-dUTP) nucleotide.
• A probe is labeled with biotin at its 5' end, preferably complementary in the middle of the gene to increase specificity.
• Streptavidin, which has high affinity to biotin, is coated in a microplate.
• Double-stranded DNA is singled by heat shock and added to the microplate.
• A complementary sequence connects to the probe, and extra material is washed with PBST.
• Anti-DIG antibody is added, and optimal density is measured by spectrophotometry.
• This method is quick and easy, providing results in a maximum of 4 hours without advanced laboratory skills, and is considered safe and non-mutagenic.
• Detection of biotinylated DNA uses an anti-DIG peroxidase conjugate with substrate ABTS to form a blue-green color reaction, visible and measurable using a spectrophotometer.

Advantages of PCR-ELISA

• More sensitive than agarose gel electrophoresis due to colorimetric analysis.
• Reduces the risk of toxicity from color materials and DNA pollution.
• High assay specificity because detection uses gene-specific probes.
• Suitable for largescale screening with standard laboratory equipment.
• Shorter analytical time compared to conventional PCR methods.
• Lower cost makes it a good alternative to quantitative PCR (qPCR) when high sensitivity is not required.

Primers used in the Test

• This GALT alleles can amplify using three sets of primers: IVS2-F, S135L-R, GALT 6-5, GSINT7R, GALT 9-5, and INTJR.

Results

• Hybridized products develop a green color after incubation with peroxidase substrate.
• This method detects the seven most common GALT gene mutations: Q188R, N314D, S135L, L195P, Y209C, IVS2, and K285N.

Interpretation of Results

• GALT genotypes are compared among mother, father, and proband following multiplex PCR of their GALT DNA.
• The proband has a mutation in K285N based on initial PCR-based analysis.
• The mother has a mutation for N314D.
• The father has mutations for both K285N and N314D.

Galactosemia is an autosomal recessive disorder caused by a deficiency in GALT. Neonates present with vomiting and diarrhea soon after milk intake. The main treatment is elimination of dietary galactose, but it doesn't prevent all complications.