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Reducing the False Positive for Guanidinoacetate Methyltransferase Enzyme Deficiency in Newborn by the Addition of a Second Tier UPLC-MS/MS Test

Biochemical/Metabolic and Therapeutics
  • Primary Categories:
    • Metabolic Genetics
  • Secondary Categories:
    • Metabolic Genetics
Introduction:
Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Newborn screening for GAMT was recently initiated in California using a two-tier approach for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). GUAC is a specific disease marker for GAMT. A routine flow injection analysis tandem mass spectrometry (FIA-MS/MS) screening method for GAMT produces false positive due to potential interfering compounds. A tier-2 ultra-performance liquid chromatography (UPLC)-MS/MS derivatized method was developed and validated to simultaneously test GAMT biomarkers in newborn specimens with abnormal results above the tier-1 screening cutoff. UPLC-MS/MS is highly sensitive and specific to separate GAMT biomarkers from potential interfering compounds, greatly improves the test performance and significantly reduces the false positive rate.

Methods:
Testing on DBS for GAMT biomarkers was conducted by first-tier screening by FIA-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. Samples above the tier-1 cutoff were then tested by a more specific tier-2 ultra-performance liquid chromatography UPLC-MS/MS method to separate GAMT biomarkers from potential isobaric interfering compounds in MRM positive ion mode with a total run time of 3.5 minutes per sample.

Results:
In total, we tested 103,124 newborns in the first three months of GAMT screening using mass spectrometry in California. Of these newborn, 544 (0.53%) newborns were screen indeterminate (GUAC≥3 µmol/L and Ratio= GUAC/Creatine ≥6) for GAMT by the tier-1 screening. Samples that did not have a positive result for both markers were screen negative. All 544 indeterminate newborn samples were tested by a tier-2 method; of these, 28 (5.15%) newborns were screen positive (GUAC≥5 µmol/L) and 516 were screen negative for GAMT (GUAC<5 µmol/L). Therefore, tier-2 method eliminates 95% of the false positive samples submitted for 2nd tier testing. Out of 28 referred newborns for evaluation – 92.8% were in the NICU at the time of specimen collection, and they all received total parenteral nutrition (TPN). Remaining referred cases - 1 newborn from the regular nursery and 1 newborn from the NICU was not on TPN. 25 (89.3%) out of 28 newborn’s GUAC tier-2 value was <7 µmol/L and 14 of these have no evidence of GAMT deficiency per clinical resolution (12 No Disorder and 2 Could not be determined), 11 are still pending resolution. The rest 3 (11%) newborn’s GUAC tier-2 was >7 µmol/L, the clinical resolution is not determined yet. No cases of GAMT deficiency were confirmed from this first 3 months of testing.

Conclusion:
Population screening using a two-tier approach is highly sensitive for the early identification of patients with GAMT disorder. Two-tier testing can help to minimize false positives and increase positive predictive value. Based on the review of screen-positive cases with tier-2 GUAC<7 µmol/L show that 14 cases were resolved negative for GAMT deficiency, and the remaining 11 pending cases are highly likely to be negative for GAMT deficiency. This makes a strong case for changing tier-2 GUAC cutoff to ≥7 µmol/L, that could reduce the false positives significantly from 5.15% to under 1%.  Providing accurate GAMT biomarkers results will help to address some of the difficult cases with faster and better “Screening to Diagnosis” turnaround time.

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