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Introduction Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited metabolic disorder that impairs the synthesis of creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. Here we report the first two cases of GAMT deficiency identified at birth by newborn screening (NBS) in Utah and New York. Methods NBS dried blood spots were analyzed by tandem mass spectrometry (MS/MS) using either derivatized or non-derivatized assays to detect guanidinoacetate (GUAC) and CRE. For any positive samples, a second-tier test using a more selective method, ultra-performance liquid chromatography (UPLC) combined with MS/MS, was performed to separate GUAC from potential isobaric interferences. Results NBS for GAMT deficiency began in Utah on June 1, 2015 using a derivatized method for the detection of GUAC and CRE. In May 2019, the laboratory and method transitioned to a non-derivatized method. GAMT screening was added to the New York State NBS panel on October 1, 2018 using a derivatized method. In New York, a total of 537,408 babies were screened, 23 infants were referred and one newborn was identified with GAMT deficiency. In Utah, a total of 273,902 infants were screened (195,425 with the derivatized method, 78,477 with the non-derivatized method), three infants referred and one was identified with GAMT deficiency. Mean levels of GUAC and CRE were similar between methods (Utah derivatized: GUAC = 1.20 ± 0.43 μmol/L, CRE = 238 ± 96 μmol/L; Utah non-derivatized: GUAC = 1.23 ± 0.61 μmol/L, CRE = 344 ± 150 μmol/L, New York derivatized: GUAC = 1.34 ± 0.57 μmol/L, CRE = 569 ± 155 μmol/L). With either Utah method, similar concentrations of GUAC are observed in first (collected around 1 day of age) and the second NBS specimens (routinely collected at 7–16 days of age), while CRE concentrations decreased in the second NBS specimens. Both infants identified with GAMT deficiency started therapy by 2 weeks of age and are growing and developing normally at 7 (Utah) and 4 (New York) months of age. Conclusions Newborn screening allows for the prospective identification of GAMT deficiency utilizing elevated GUAC concentration as a marker. First-tier screening may be incorporated into existing methods for amino acids and acylcarnitines without the need for new equipment or staff. Newborn screening performed by either derivatized or non-derivatized methods and coupled with second-tier testing, has a very low false positive rate and can prospectively identify affected children. SummaryCerebral creatine deficiency syndromes caused by defects in creatine synthesis can result in intellectual disability, and are preventable if therapy is initiated early in life. This manuscript reports the identification of two infants with GAMT deficiency (one of the cerebral creatine deficiency syndromes) by newborn screening and demonstrates NBS feasibility using a variety of methods.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder that results in reduced activity of guanidinoacetate methyltransferase, an accumulation of guanidinoacetate (GUAC), and a lack of cerebral creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. If started early in life, treatment with creatine supplements is highly effective. Because there are reliable biomarkers for GAMT deficiency, GUAC and CRE, and because the disorder is readily treatable with a significant improvement in outcomes, GAMT deficiency is an excellent candidate for newborn screening. Several programs have conducted pilot programs or started screening. An isobaric interferant of the GUAC marker has been reported which may cause false positive results. To reduce the number of false positives, a second-tier HPLC test to separate GUAC from unknown, isobaric interferants may be incorporated into the screening algorithm. New York State began screening for GAMT deficiency in October 2018 using a three-tiered screening approach. Quantification of GUAC and CRE were incorporated into routine screening for amino acids and acylcarnitines. In the first year of screening a total of 263,739 samples were tested for GAMT deficiency. Of these, 3382 required second tier testing. After second tier testing, 210 repeat specimens were requested for borderline results and 10 referrals were made to specialty care centers for confirmatory testing. In the first year of screening there were no confirmed cases of GAMT deficiency detected. To reduce the number of samples needing second tier testing and the number false positives we explored the use of a second MS transition to confirm the identity of the GUAC marker. GUAC and its internal standard are detected as butylated esters after sample preparation and derivatization. The original method used transition of the GUAC molecular ion of m/z 174.1 to a reactant ion of m/z 101.1. To confirm the identity of the GUAC marker we selected a qualifier ion of 174.173. The alternative product ion results were found to agree more closely with the second tier HPLC-MS/MS results for GUAC. It was found that the alternative transition may be used for quantification of the GUAC marker with acceptable analytical performance (linearity, accuracy, and precision). On March 5, 2020, the method of analysis for GUAC was modified to use the alternative product ion. For a comparable 6-month period, the modified method reduced the number of samples requiring second tier testing by 98%, reduced the number of borderline results requiring a repeat sample by 87.5%, and reduced the number of referrals to specialty care centers by 85%. Using the modified method, the correlation (r-squared) of the first and second tier screening results for GUAC is greater than 0.95. Since the first-tier results correlate well with the second-tier results, the second-tier screening is no longer necessary with the modified method.

Objective The aim of this study was to develop a newborn screening algorithm for Krabbe disease. Design and methods We measured the galactocerebrosidase activity of 139,074 anonymous newborns, 56 known carriers, and 16 Krabbe patients using a tandem mass spectrometry method. The activities were converted to percentages of daily mean activity (%DMA), and the results from diseased and normal populations were used to establish cutoffs. Results The absolute activities for the newborns ranged from 0.17 to 355 μmol/L h ( N = 139,074) and activities for Krabbe-positive controls ranged from 0.08 to 0.48 μmol/L h ( N = 16, n = 91 measurements) while activities for carriers ranged from 0.28 to 2.71 μmol/L h ( N = 56, n = 72 measurements). Cutoffs were set based on results from Krabbe-positive and carrier controls and the newborn population distribution. Conclusions The algorithm and cutoffs we propose provided 100% detection of all positive controls with 60/100,000 screen positive results predicted. In the course of this study, one anonymous newborn was predicted to have Krabbe disease based on enzyme activity and subsequent DNA analysis.