LC–MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers

Objective: Heterozygosity in 21-hydroxylasedeficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry. Design: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared. Method: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC‐MS/MS) in 58 carriers (35 males, 23 females, age range 6‐78 years) and 44 random controls (25 males, 19 females, age range 8‐58 years). Results: Heterozygotes could be identified best applying the 17-hydroxyprogesteroneC21-deoxycortisol/cortisol!1000 ((17OHPC21S)/F!1000) equation 30 min after ACTH injection. An optimal cut-offvalue of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-offis 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%. Conclusion: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHPC21S)/ F!1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC‐MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-offand to a specificity of even 100% applying a cut-offhigher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHPC21S)/F!1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism.