Enhancing newborn screening for tyrosinemia type I.

Hepatorenal tyrosinemia, also known as tyrosinemia type I (Tyr-I) is an autosomal recessive inborn error of metabolism. The primary enzyme defect has been attributed to a deficiency of fumarylacetoacetase (EC 3.7.1.2) (1 ). Tyr-I is usually asymptomatic in newborns, but if left untreated it affects liver, kidney, bone, and peripheral nerves; in its most severe form, affected infants may die from liver failure in the first months of life (2 ). The majority of Tyr-I patients can be treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) with promising outcomes (3, 4 ). The incidence of this condition is estimated to be 1:100 000 but may be much higher in certain populations such as the province of Quebec, where it occurs once in every 20 000 live births (5 ). Given the effectiveness of treatment and a test for its marker in the Guthrie specimen, Tyr-I fulfills the criteria for newborn screening and is included in the American College of Medical Genetics’ core panel of newborn screening recommendations (6 ). Most newborn screening programs detect Tyr-I by measurement of tyrosine in Guthrie specimens (dried blood spot) (7–9 ). This method is convenient because tyrosine can be extracted and monitored along with other amino acids and acylcarnitines present in newborn screening panels (7–9 ). It has become clear, however, that tyrosine alone is an inadequate marker for screening of Tyr-I because tyrosine measurement lacks specificity and cannot distinguish Tyr-I from other disorders in tyrosine catabolism and transient hypertyrosinemia (10, 11 ). Even more disconcerting are reports of missed cases of Tyr-I in newborns with either normal or only modestly increased concentrations of tyrosine (9, 12–14 ). A number of recent publications have reported newborn screening results for tyrosine at or near the population average for confirmed cases of Tyr-I (9, 12–14 ). Cases have been reported with tyrosine concentrations of approximately 120 mol/L, and one case was reported with a tyrosine concentration of approximately 50 mol/L. Data from New York State Newborn Screening show the average concentration of tyrosine in Guthrie specimens in the New York State population is approximately 85 mol/L. If tyrosine is used as the only marker for Tyr-I, it is very likely that cases will be missed. Increased concentrations of succinylacetone (SUAC) are characteristic of Tyr-I (2 ). A method for the analysis of SUAC in Guthrie specimens has been reported (13, 14 ). This assay was demonstrated to be suitable for newborn screening in that it is sensitive, selective, simple, and amenable to processing high numbers of samples. SUAC can be extracted from residual specimen punches that were extracted previously for amino acids and acylcarnitines, and the SUAC is then quantified by tandem mass spectrometry. Because this SUAC measurement requires a separate extraction and analysis from the amino acid/ acylcarnitines analysis, however, this method would essentially double the number of samples to be run daily on a laboratory’s mass spectrometer. It may be possible to implement the extraction and analysis of SUAC as a second-tier test of only samples with increased tyrosine, but the cutoff concentration for tyrosine would have to be set low, so a significant number of samples would need to be analyzed. In New York State, we estimate that a cutoff of 180 mol/L for tyrosine would result in SUAC analysis of approximately 20 samples per day, and a cutoff of 120 mol/L would result in 125 samples per day. Even then, it is still likely that cases of Tyr-I would be missed. Since December 2007, this program has tested all specimens for SUAC using the method described by Allard et al. (13 ). To date, SUAC analyses have not identified a Tyr-I specimen. In this issue of Clinical Chemistry, Turgeon et al. (15 ) report a combined assay for SUAC, amino acids, and acylcarnitines that does not require any additional mass spectrometer instruments. In this assay, punches from the Guthrie specimen are first extracted using a methanol solution containing amino acid, carnitine, and acylcarnitine internal standards (ISs) (7–9 ). The methanol extract is removed and transferred to another microtiter plate for butylation of the amino acids and acylcarnitines, and the residual blood spots are left in place. SUAC is extracted from these spots in an acetonitrile:water IS solution containing hydrazine. After extraction of SUAC from the leftover spots, the eluates are transferred to another round-bottom 96well plate. The butylated sample extract derived from each sample is added to the corresponding well in the plate containing the SUAC-hydrozone/IS residue, thereby combining the amino acid/acylcarnitine and SUAC extracts into a single well. Tandem mass spectrometry analysis is performed for SUAC by selected reaction monitoring (SRM) experiments added to the precursor, neutral loss, and SRM scans for amino acids, carnitine, and acylcarnitines (7–9 ). In this and other Clinical Chemistry 54:4 627–629 (2008) Editorial