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Discriminating Guanidinoacetate Methyltransferase Enzyme Deficiency false positives TPN babies by using the Arginine/GUAC ratio

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). A routine flow injection analysis tandem mass spectrometry (FIA-MS/MS) screening method for GAMT uses cut-off of GUAC≥3 µmol/L and Ratio= GUAC/Creatine ≥6. 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. Samples with tier-2 GUAC result ≥5 µmol/L were considered positive, and a referral was made to a specialty area service center. Herein we review the results of the first 100,000 samples tested from July to October 2024. In this study, newborn with tier-2 GUAC result ≥5 µmol/L were further examined for Arginine to GUAC ratio.

Methods:
Testing on DBS for GAMT biomarkers was conducted by tier-1 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. We conducted a review of our screening algorithm, referral rates and clinical outcomes for screen-positive newborns.

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%) newborns were screen positive (GUAC≥5 µmol/L) and 516 (95%) were screen negative for GAMT (GUAC<5 µmol/L). 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. All 28 referred cases were further examined for Arginine to GUAC Ratio. The GUAC range was 5.05 – 35.8 µmol/L, Arginine range was 19 – 512 µmol/ and Arginine/GUAC ratio was 3.38-47.33.  The Arginine values from these 28 cases were significantly higher than compared to the 516 newborns with tier-2 normal GUAC values. We further compared the results from our confirmed GAMT case with GUAC value of 16.62 µmol/L, Arginine value of 10.00 µmol/L and Arginine/GUAC ratio=0.60. This ratio is significantly lower than the ratio from 28 referred cases.

Conclusion:
Based on the review, the Arginine/GUAC ratio was significantly higher in unaffected newborns with elevated GUAC values (Ratio=3.38 to 47.33) compared to newborn with GAMT deficiency (Ratio=0.60). This ratio could be used in conjunction with tier-2 elevated GUAC to further differentiate false positive cases from true GAMT deficiency cases. 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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