Expanded Steroid Profiling Identifies Novel Newborn Screening Markers for Congenital Adrenal Hyperplasia

Abstract Context Simultaneous measurement of multiple steroids in dried bloodspots facilitates accurate newborn screening (NBS) for classic congenital adrenal hyperplasia (CAH), although false-positive (FP) tests are encountered. Some babies with milder forms of CAH, who may benefit from early detection and treatment, can be missed by screening. Objective This work aims to evaluate a method for expanded steroid profiling in NBS bloodspots to identify novel sensitive and specific markers that will inform improvements for screening for CAH, and to identify the metabolic routes to excessive androgen synthesis in CAH in the newborn period. Methods A method to measure 41 steroids by liquid chromatography–tandem mass spectrometry is described and evaluated using manufactured bloodspots and residual NBS specimens from 43 babies with true-positive (TP), 11 babies with false-negative (FN), and 389 babies with false-positive (FP) screening results. Mann-Whitney analysis was used to determine if steroid measurements could distinguish between samples from babies with CAH and FP specimens. Results The method was sufficiently precise and accurate for all steroids but was less sensitive for steroids of the mineralocorticoid pathway. Seven novel 11-oxygenated steroid markers were higher in TP and FN samples when compared to the FP group and were the most informative for screening. The most sensitive and specific marker, 21-deoxycortisone, was elevated in 52 of 53 CAH cases and was not detected in any FP specimens. Conclusion The 11-oxygenated steroids are the most sensitive markers in dried bloodspots in the newborn period. Steroid profiling suggests that androgen excess in CAH in the newborn period is via several interconnected metabolic routes.