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

Abstract Background Simultaneous measurement of multiple steroids in dried bloodspots facilitates accurate newborn screening for classical CAH, although false positive tests are encountered. Some babies with milder forms of CAH, who may benefit from early detection and treatment, can be missed by screening. Objective To evaluate a method for expanded steroid profiling in newborn screening 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 LCMSMS is described and evaluated using manufactured bloodspots and residual newborn screening 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. A most sensitive and specific marker, 21-deoxycortisone, was elevated in 52 of 53 CAH cases and was not detected in any FP specimens. Conclusions 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.