Method modification to reduce false positives for newborn screening of guanidinoacetate methyltransferase deficiency.

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 174.1 > 73. 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.

A surprising treatment response in a patient with rare isolated growth hormone deficiency, type IB.

Isolated Growth Hormone Deficiency (IGHD) is a rare cause of short stature, treated with the standard regimen of subcutaneous synthetic growth hormone (GH). Patients typically achieve a maximum height velocity in the first year of treatment, which then tapers shortly after treatment is stopped. We report a case of a 9-year-old male who presented with short stature (<3rd percentile for age and race). Basal hormone levels showed undetectable serum IGF1. Skeletal wrist age was consistent with chronologic age. Cranial MRI revealed no masses or lesions. Provocative arginine-GH stimulation testing demonstrated a peak GH level of 1.4 ng/mL. Confirmatory genetic testing revealed a rare autosomal recessive single-nucleotide polymorphism (SNP) with mutational frequency of 2%. GH supplementation was started and pursued for 2 years, producing dramatically increased height velocity. This velocity persisted linearly through adolescence, several years after treatment had been discontinued. Final adult height was >95th percentile for age and race. In conclusion, this is a case of primary hypopituitarism with differential diagnosis of IGHD vs Idiopathic Short Stature vs Constitutional Growth Delay. This case supports two objectives: Firstly, it highlights the importance of confirmatory genetic testing in patients with suspected, though diagnostically uncertain, IGHD. Secondly, it demonstrates a novel secondary growth pattern with implications for better understanding the tremendous variability of GH treatment response. LEARNING POINTS: GHD is a common cause of growth retardation, and IGHD is a specific subtype of GHD in which patients present solely with short stature.The standard treatment for IGHD is subcutaneous synthetic GH until mid-parental height is reached, with peak height velocity attained in the 1st year of treatment in the vast majority of patients.Genetic testing should be strongly considered in cases of diagnostic uncertainty prior to initiating treatment.Future investigations of GH treatment response that stratify by gene and specific mutation will help guide treatment decisions.Response to treatment in patients with IGHD is variable, with some patients demonstrating little to no response, while others are 'super-responders.'