Androgen Excess and Diagnostic Steroid Biomarkers for Nonclassic 21-Hydroxylase Deficiency Without Cosyntropin Stimulation

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A F Turcu and others Steroid biomarkers in N21OHD 183:1 63–71 Clinical Study

Androgen excess and diagnostic steroid biomarkers for nonclassic 21-hydroxylase deficiency without cosyntropin stimulation

Adina F Turcu1, Diala El-Maouche2, Lili Zhao3, Aya T Nanba1, Alison Gaynor2, Padma Veeraraghavan2, Richard J Auchus1,4 and Deborah P Merke2,5

1Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan, USA, 2National Correspondence Institutes of Health (NIH) Clinical Center, Bethesda, Maryland, USA, 3School of Public Health, 4Department of should be addressed Pharmacology, University of Michigan, Ann Arbor, Michigan, USA, and 5Eunice Kennedy Shriver National Institute of to A F Turcu Child Health and Human Development, Bethesda, Maryland, USA Email [email protected]

Abstract

Objectives: The clinical presentation of patients with nonclassic 21-hydroxylase deficiency (N21OHD) is similar with that for other disorders of androgen excess. The diagnosis of N21OHD typically requires cosyntropin stimulation. Additionally, the management of such patients is limited by the lack of reliable biomarkers of androgen excess. Herein, we aimed to: (1.) compare the relative contribution of traditional and 11-oxyandrogens in N21OHD patients and (2.) identify steroids that accurately diagnose N21OHD with a single baseline blood draw. Design: We prospectively enrolled patients who underwent a cosyntropin stimulation test for suspected N21OHD in two tertiary referral centers between January 2016 and August 2019. Methods: Baseline sera were used to quantify 15 steroids by liquid chromatography-tandem mass spectrometry. Logistic regression modeling was implemented to select steroids that best discriminate N21OHD from controls. Results: Of 86 participants (72 females), median age 26, 32 patients (25 females) had N21OHD. Age, sex distribution, and BMI were similar between patients with N21OHD and controls. Both testosterone and androstenedione

European Journal of Endocrinology were similar in patients with N21OHD and controls, while four 11-oxyandrogens were significantly higher in patients with N21OHD (ratios between medians: 1.7 to 2.2, P < 0.01 for all). 17α-Hydroxyprogesterone (6.5-fold), 16α-hydroxyprogesterone (4.1-fold), and 21-deoxycortisol (undetectable in 80% of the controls) were higher, while corticosterone was 3.6-fold lower in patients with N21OHD than in controls (P < 0.001). Together, baseline 17α-hydroxyprogesterone, 21-deoxycortisol, and corticosterone showed perfect discrimination between N21OHD and controls. Conclusions: Adrenal 11-oxyandrogens are disproportionately elevated compared to conventional androgens in N21OHD. Steroid panels can accurately diagnose N21OHD in unstimulated blood tests.

European Journal of Endocrinology (2020) 183, 63–71

Introduction

Congenital adrenal hyperplasia comprises a series of in girls and life-threatening adrenal insufficiency. autosomal recessive defects in enzymes required for cortisol Patients with milder enzymatic defects, conventionally biosynthesis. The most common form of congenital termed ‘nonclassic’ or ‘late-onset’ 21OHD, have normal adrenal hyperplasia is 21-hydroxylase deficiency (21OHD) glucocorticoid and mineralocorticoid production, owing (1). In its most severe forms, also called ‘classic’, 21OHD to a compensatory increase of corticotropin (ACTH) typically presents at birth, with virilized external genitalia secretion. The increased ACTH stimulation, however,

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-20-0129 European Journal of Endocrinology https://eje.bioscientifica.com quantified fromasmall-volume aliquotofserum. With the (LC-MS/MS) assays,multiplesteroidscanbe spectrometry 21OHD arelacking. classic 21OHD ( correlate well with surrogates of poor disease control in with classic21OHD( C 11-ketotestosterone (11KT) and the other 11-oxygenated and othershaveshownthatthepotentandrogen inthese patients.Weof hyperandrogenismobserved do notalwaysaccountfortheclinicalmanifestations are oftennormalinpatientswithnonclassic21OHDand elevated inpatientswithclassic21OHD,thesesteroids ( and 11 amounts of11 androstenedione (A4) andtestosterone, leading toample 11 of theabundantlyaccumulated17OHP4.Similarly, 18 lyase (CYP17A1)and21-deoxycortisol(21dF)( normally aminorproductof17 16 accretion ofother21-carbon(C immunoassays ( women withirregularmenses,andwhenmeasuredby so inprematureandsicknewborns,reproductive-age that false-positiveelevationsarecommon,particularly An additionallimitationof17OHP4screeningtestingis diagnostic threshold of 1000 ng/dL (30 nmol/L) ( at screening( (cosyntropin) stimulation,when17OHP4iselevated requires dynamic testing with synthetic ACTH In contrast, the diagnosis of nonclassic 21OHD usually by newbornscreeninginmostdevelopedcountries( elevations inrandomsamplesandaretypicallydiagnosed ( 17 3 similar tothatforpolycysticovariansyndrome(PCOS)( clinical phenotypeofwomenwithnonclassic21OHDis with other disorders of androgen excess. In particular, the menses, andinfertilityormiscarriages,featuresshared such asprematurepubarche, hirsutism,acne,irregular nonclassic 21OHDpresentwithclinicalmanifestations to excessadrenalandrogenproduction.Patientswith in conjunctionwiththe21-hydroxylasedefect,leads 1 20 , 19 ). Patientswithclassic21OHDhavemarked17OHP4 Clinical Study 4 β , α α steroids (11-oxyandrogens) are elevated in patients , -hydroxylase (CYP11B1)canalsoutilizeassubstrates -hydroxyprogesterone (16OHP4)( -hydroxyprogesterone (17OHP4)measurements , Using liquidchromatography-tandem mass Beyond 17OHP4,21OHDpromotesthe The diagnosis of 21OHD relies on serum 5 21 19 ). ). WhileestosteroneandA4arealsofrequently ), whichresultsfromthe11 β -hydroxytestosterone (11OHT),respectively > 200 ng/dL,6nmol/L)butnotabovethe 10 24 β , ), but data in patients with nonclassic -hydroxyandrostenedione (11OHA4) 11 , 12 22 , , 13 23 , 14 ). The11-oxyandrogens A FTurcuandothers , 15 21 α ) steroids,suchas ). -hydroxylase/17,20- 16 β -hydroxylation ), whichis 7 16 , 8 , , 1–24 17 9 6 2 ). ). , ,

conducted in the morning, in outpatient setting. The August 2019wereincludedinthisstudy. Alltestswere 2016 and suspected nonclassic 21OHD between January and theNationalInstitutesofHealthClinicalCenter)for referralcenters(UniversityofMichigan in twotertiary hyperandrogenism with cosyntropinstimulationtesting Patients undergoingclinicalevaluationfor Study participants Patients andmethods blood draw. diagnose nonclassic21OHDreliablywithasinglebaseline these patientsand(2.)toidentifysteroidpanelsthatcan of traditionalandadrenal-specific11-oxyandrogensin a dual goal: (1.) to compare the relative contribution in patientswithnonclassic21OHDofbothsexes, current study, weaimedtoassessthesteroidfingerprints Spearman’s correlation coefficients. Receiver-operating groups. Correlations between steroids were assessed using test wasusedtocompare sexdistributionsbetween comparisons of continuous variables,and Chi-square Mann–Whitney Statistical analyses performed byLC-MS/MS,aspreviouslydescribed( 11-oxyandrogens, from sera obtained atbaseline was cortisol precursors,testosterone,A4,andfour Quantitation of15unconjugatedΔ Steroid quantitationbymassspectrometry standard clinicalcare. by theIRBforusingleftoverserumcollectedaspartof University ofMichigan,awaiverconsentwasgranted 8 years old, gave written assent. For patients seen at the NIH providedinformedconsent,andallminors,atleast Adult patients andparents of participating minors seen at approval fromeachofthetwoparticipatinginstitutions. were conductedwithInstitutionalBoardReview(IRB) Androgen ExcessandPCOSSocietycriteria( the diagnosis.ThediagnosisofPCOSwasbasedon studies werealsoperformedandconsistentwith ng/dL (300nmol/L).Inallbutthree21OHDcases,genetic concentrations of on clinical phenotype and stimulated serum 17OHP4 diagnosis ofnonclassic21OHDwasdeterminedbased Steroid biomarkersinN21OHD > U 1000 ng/dL(30nmol/L)and -test wasusedfortwo-group Downloaded fromBioscientifica.com at09/29/202106:56:02PM 4 183 steroids,including :1 25 ). Allstudies < 26 10 000 ). 64 via freeaccess European Journal of Endocrinology 21-hydroxylase deficiency;PCOS,polycystic ovarysyndrome. AI, adrenalinsufficiency;F,females; M,males;N21OHD,nonclassic details available. suspicion ofcongenitaladrenalhyperplasia (CAH),withoutotherclinical *Patients referredfromoutsidefor cosyntropin stimulationwiththe Suspicion for Early onset Short stature Suspicion forAI Precocious Menstrual Hirsutism PCOS Diagnosis ( BMI (kg/m Sex (F/M) Age (years) n and interquartilerangeforBMI. participants. Dataareexpressedasmediansandrangeforage Table 1 were similarinpatientswithnonclassic21OHDandthose Of theC or withoutnonclassic21OHD Comparison ofandrogensbetweenpatientswith vs thosewithout21OHD. distribution, andBMIweresimilarbetweenpatientswith (50%) patientsmetcriteriaforPCOS.Overall,age,sex stimulated 17OHP4values females), 21OHDwasexcludedbasedoncosyntropin- 21OHD ( these, 32patients(25females)metcriteriafornonclassic and 70 years, median age 26, participated in this study. Of In total,86patients(72females),withagesbetween6 Results from controls. that bestdiscriminatepatientswithnonclassic21OHD implemented fortheselectionofasmallsetsteroids due tohighcorrelationbetweenbiomarkers),was penalty, combinedwithclinicalknowledge(relevant prediction performance.Logisticregressionwithalasso wereusedtocharacterizethe characteristics (ROC)curves Clinical Study CAH* alopecia pubarche dysfunction Demographic andclinicalcharacteristicsofstudy 19 2 Table 1 n ) steroidsmeasured,bothtestosteroneandA4 ) ). Intheremaining54patients(47 27.9 (23.3–32.9) 27 (6–70) Controls 47/7 27 54 4 1 1 4 5 3 9 < 30 nmol/L.Ofthelatter, 27 A FTurcuandothers 25.1 (20.6–29.7) 25 (6–66) N21OHD 25/7 32 0.085 0.367 0.223 P 11 and controls.11KT,11-ketotestosterone; 11OHA4, patients withnonclassic21-hydroxylase deficiency(N21OHD) Comparison oftraditionaland 11-oxyandrogens between Figure 1 when comparingadultwomenwithnonclassic21OHD P 21OHD (from2.1-foldfor11KA4to2.9-fold11KT, patients with nonclassic 21OHD than in those without four 11-oxyandrogens were significantly higherinfemale younger thantheirunaffectedcounterparts( 21OHD, whilemalepatientswithnonclassic21OHDwere between femalepatientswithandwithoutnonclassic groups ( contrast, theA4/Tratiowassimilarbetweentwo than in unaffected individuals ( significantly higherinpatientswithnonclassic21OHD and 11OHA4/A4(2.6(2.1–4.6)vs1.2(0.7–2.1))were 2 to 2.2-foldfor11OHT( patients withnonclassic21OHD,from1.7-foldfor11KA4 all four 11-oxyandrogens were significantly higher in without 21OHD( distribution betweenthegroups( due to the small number of patients and differences in age hormonal differencescouldnotberigorouslycompared, in otherdisordersofandrogenexcess( and apredominantovariansource oftestosteroneandA4 androgens infemalepatientswithnonclassic21OHD suggesting apredominantadrenaloriginforallthese patients withnonclassic21OHDthanthoseunaffected, testosterone and A4, respectively, were tighter in female between 11KTand11OHA4withtheirprecursors, with PCOSwomen( Steroid biomarkersinN21OHD

). Furthermore, 11KT/T (1.8(1.2–3.0)vs0.6(0.2–0.9)) < β

-hydroxyandrostenedione. 0.0001 forall, In asubgroupanalysis by sex, age and BMI weresimilar P =0.3). P Table 3

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0.001 for both). In Fig. 2 :1 ). Fig. 1 Table 3 ). Inmales, and Table ). All 65 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com CI: 79.19–99.23%). (95% CI:87.25–99.55%)and aspecificityof93.75(95% A 17OHP4of5.68nmol/L showed asensitivityof96.30 when usedinisolation,with anAUCof0.986( power between thetwogroups excellent discriminatory (95% CI: 83.78–99.92%). 17OHP4 also demonstrated (95% CI,CI:87.25–99.55%)andaspecificityof96.88 and 21dFvalues (AUC)whenusedalone (0.997, under thecurve Of allsteroidsmeasured,21dFshowedthehighestarea and corticosterone( showed thatcombined17OHP4( and 19 of these were female. Logistic regression modeling a 17OHP4serumconcentration ( showed no statistical difference between the two groups cortisol, 11-deoxycortisol,and11-deoxycorticosterone lower in patients with nonclassic 21OHD ( ( undetectable in80%ofthecontrols, (6.5-fold), 16OHP4(4.1-fold),and21-deoxycortisol(21dF, for:17OHP4 nonclassic 21OHDvscontrolswasobserved The highestrelativedifferencebetweenpatientswith or withoutnonclassic21OHD Comparison ofC 11-ketotestosterone. 11dF, 11-deoxycortisol;11OHA4,11 A4, androstenedione;DOC,11-deoxycorticosterone;17OHP4,17 A4/Testosterone 11OHA4/A4 11KT/Testosterone 11KT 11OHT 11KA4 11OHA4 Testosterone A4 DOC 11dF Corticosterone Cortisone Cortisol Progesterone 21dF 16OHP4 17OHP4 Steroid controls. Dataareexpressedasmedian(interquartilerange). Table 2 Table 2 Table 2 Clinical Study Of 32patientswithnonclassic21OHD,23(72%)had (nmol/L) ). ). Conversely, corticosteronewassignificantly Comparison ofsteroidconcentrationsbetweenpatientswithnonclassic21-hydroxylasedeficiency(N21OHD)and > 21 0.64 nmol/Lhadasensitivityof96.30 β steroidsbetweenpatientswith 3 = − 0.43) hadanAUCof1( β -hydroxyandrostenedione; 11KA4,11-ketoandrostenedione; 11OHT11 202.5 (125.9–442.8) 53.7 (30.5–72.3) β A FTurcuandothers 3.8 (2.8–4.5) 2.6 (2.1–4.6) 1.8 (1.2–3.0) 1.8 (1.2–3.3) 1.0 (0.3–1.7) 1.1 (0.7–1.7) 9.6 (5.1–25.9) 1.1 (0.5–2.7) 4.3 (2.0–8.1) 0.3 (0.3–0.4) 0.8 (0.5–1.4) 1.6 (0.8–3.5) 0.8 (0.5–1.4) 1.8 (1.3–5.3) 2.4 (1.4–4.9) 1.1 (0.5–2.7) < N21OHD 1 0.05), 21dF ( 21dF =0.05), 30 nmol/Latbaseline P ( P n

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< < Table 5 2 α =1.45), 0.001). 0.001), -hydroxyprogesterone; 16OHP4,16-hydroxyprogesterone;21dF,21-deoxycortisol; 249.1 (208.9–373.7) 51.5 (40.3–60.8) ). 3.5 (2.4–4.2) 1.2 (0.7–2.1) 0.6 (0.2–0.9) 0.9 (0.6–1.3) 0.4 (0.3–0.6) 0.6 (0.4–0.8) 4.5 (2.9–7.1) 1.6 (0.8–4.3) 3.9 (2.2–8.5) 0.5 (0.4–0.9) 0.9 (0.4–1.6) 5.9 (3.5–13.6) 0.4 (0.3–0.8) 0.6 (0.4–0.9) 1.6 (0.8–4.3) Controls 0 (0–0.3) for simultaneouselevations of21dFand17OHP4plus immunoassay-based 17OHP4 alone,LC-MS/MStesting differential diagnostictesting. nonclassic 21OHDamongchildrenandadultsundergoing samples showed the highest accuracy in diagnosing and corticosteronemeasuredinunstimulatedserum assay, we found that the combination of 17OHP4, 21dF, androgen excess. Secondly, using a 15-steroid LC-MS/MS 21OHD as compared to patients with other disorders of than testosteroneandA4inpatientswithnonclassic found that11-oxyandrogensaredisproportionatelyhigher 11-oxyandrogens in patientswithnonclassic 21OHD, we blood drawsordynamictesting.Inthisfirststudy of decision-making andcircumvent theneedforadditional biospecimens. Multiple-steroidassayscouldassistclinical multiple steroidsinasingleassay, usingsmall-volume 31 assays, whicharehighlysensitiveandspecific( transitioned to steroid quantitation by mass spectrometry of similarsteroids( of sensitivity and in the presence of high concentrations can yieldunreliableresults,particularlynearthelimits immunoassays, whicharelimitedbycross-reactivityand Since the1960s,steroidshavebeenmeasuredby Discussion Steroid biomarkersinN21OHD ). Inaddition,LC-MS/MSalsoaffordsquantitationof ( n Compared toconventional newborn screeningusing = 54) Ratio ofmediansN21OHD/Controls β 27 -hydroxytestosterone; 11KT, Downloaded fromBioscientifica.com at09/29/202106:56:02PM , 28 N/A 1.3 2.0 3.3 2.0 2.2 1.7 2.1 0.7 1.1 0.6 0.8 0.3 1.0 0.8 1.8 4.1 6.5 ). Manylaboratorieshave 183 :1 < < < < < < < < 0.274 0.0001 0.0001 0.0001 0.0019 0.0001 0.0001 0.195 0.798 0.113 0.626 0.0001 0.855 0.197 0.0018 0.0001 0.0001 0.0001 29 P , 66 30 via freeaccess , European Journal of Endocrinology

Table 3 Comparison between patients with nonclassic 21-hydroxylase deficiency (N21OHD) and controls of the same sex. Data are expressed as median (interquartile range). Clinical Study

Females Males N21OHD (n = 25) Controls (n = 47) Ratio* P N21OHD (n = 7) Controls (n = 7) Ratio* P Age (years) 27 (18–34) 28 (21–36) 0.696 9 (8–13) 18 (12–52) 0.036 BMI (kg/m2) 26 (22–32) 28 (23–33) 0.224 21 (19–26) 26 (15–31) 0.62 17OHP4 (nmol/L) 16.8 (8.1–55.7) 2.3(1–4) 7.4 <0.0001 24.9 (13.6–55.7) 2.8 (2.1–3.8) 9.0 0.0006 16OHP4 (nmol/L) 2.3 (1–7.7) 0.6 (0.3–0.9) 4.1 <0.0001 2.8 (1.5–7.7) 0.6 (0.5–0.9) 5.0 0.002 21dF (nmol/L) 1.8 (1.2–5.1) 0.0 (0.0–0.0) N/A <0.0001 1.5 (1.3–5.1) 0.0 (0.0–0.5) N/A 0.0006 Progesterone (nmol/L) 0.8 (0.4–1.5) 0.4 (0.3–0.8) 1.8 0.014 1.0 (0.5–1.5) 0.4 (0.3–0.8) 2.2 0.052 Cortisol (nmol/L) 233.4 (128.1–555) 256.0 (206.6–423.6) 0.9 0.429 189.7 (81.4–222.1) 219.0 (129.4–245.4) 0.9 0.318 Cortisone (nmol/L) 57 (31.2–64.2) 52.5 (44.6–63.7) 1.1 0.823 50.4 (23.4–64.2) 39.4 (20.6–52.9) 1.3 0.710 Corticosterone (nmol/L) 2.0 (0.8–1.7) 5.4 (3.3–15.3) 0.4 <0.0001 1.4 (0.5–1.7) 5.0 (3.3–14.7) 0.3 0.0006

11dF (nmol/L) 0.8 (0.5–1.4) 0.6 (0.4–1.4) 1.2 0.482 1.1 (0.6–1.4) 1.1 (0.6–1.6) 1.0 0.477 A FTurcuandothers DOC (nmol/L) 0.3 (0.3–0.4) 0.4 (0.2–0.7) 0.8 0.322 0.3 (0.3–0.4) 0.6 (0.5–0.8) 0.5 0.001 A4 (nmol/L) 4.7 (2.6–3.4) 4.8 (3.1–7.1) 1.0 0.890 1.9 (0.6–3.4) 2.1 (1.0–2.4) 0.9 0.901 Testosterone (nmol/L) 1.2 (0.8–11.2) 1.2 (0.9–2.8) 1.0 0.696 0.4 (0.2–11.2) 9.4 (0.6–16) 0.04 0.137 11OHA4 (nmol/L) 10.1 (5.8–28.4) 4.4 (2.9–7.3) 2.3 <0.0001 5.4 (3.1–28.4) 4.2 (1.6–5.3) 1.3 0.318 11KA4 (nmol/L) 1.3 (0.7–1.4) 0.6 (0.5–0.8) 2.1 0.0003 0.9 (0.4–1.4) 0.6 (0.3–0.8) 1.4 0.105 11OHT (nmol/L) 1.1 (0.5–0.5) 0.4 (0.3–0.6) 2.6 <0.0001 0.3 (0.2–0.5) 0.3 (0.1–0.4) 1.1 0.685 11KT (nmol/L) 2.4 (1.4–2.3) 0.8 (0.6–1.3) 2.9 <0.0001 1.4 (0.9–2.3) 0.4 (0.3–1.0) 3.2 0.024

*Ratio of medians N21OHD/Controls. A4, androstenedione; DOC, 11-deoxycorticosterone; F, females; M, males; 17OHP4, 17α-hydroxyprogesterone; 16OHP4, 16α-hydroxyprogesterone; 21dF, 21-deoxycortisol; 11dF, 11-deoxycortisol; 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11OHT 11β-hydroxytestosterone; 11KT, 11-ketotestosterone. patients withnonclassic21OHDfromunaffected that baseline 17OHP4 performed well indistinguishing with nonclassic21OHD.UsingLC-MS/MS,wefound and A4concentrationsaretypicallynormalinpatients 21OHD is commonly pursued. Furthermore, serum cortisol biochemical testingtodistinguishPCOSfromnonclassic with irregular menses and/or oligomenorrhea, in whom timing, however, often becomes impractical in patients of 17OHP4anditsriseduringtheluteal phase. Accurate phase ( samples shouldideallybeobtainedintheearlyfollicular ( in randomsamplesfrompatientswithnonclassic21OHD hormonal abnormalities,including17OHP4,aresubtle elevations seeninpatientswithclassic21OHD,most for classic21OHD.Incontrasttothemarked17OHP4 found tobesuperior17OHP4fornewbornscreening ( low cortisolwasshowntoreducefalse-positiveresults hyperandrogenism inwomen withnonclassic21OHDare patients withnonclassic 21OHD ( excess. Treatment forsymptomatic istypicallyreserved is themonitoringand management ofandrogen 17OHP4 or21dFalone. in the few cases with ambiguousclassification based on added valueofsuchpanelscouldbeparticularlyrelevant the patients with nonclassic 21OHD from controls. The baseline showedthehighestaccuracyfordistinguishing Together, 17OHP4, 21dF, and corticosterone measured at the diagnosisofnonclassic21OHDhavebeenlacking. regarding theutilityof21dFasasinglebaselinetestfor for the diagnosis of 21OHD ( it hasbeenproposedasabetteralternativeto17OHP4 does nottrackwithphysiologic17OHP4elevations,and 11-deoxycortisol and subsequently to cortisol.Thus,21dF the unhinderedglucocorticoidpathwaytakes17OHP4to 11 17OHP4 isconvertedto21dFbyfullyfunctionalenzyme and specificity for nonclassic 21OHD. In 21OHD, of 0.64nmol/Ldemonstratednearlyperfectsensitivity in bothgroups.Notably, anunstimulated21dFvalue 11-deoxycortisol, andotherintermediatesweresimilar than incontrols,whilecortisol,11-deoxycorticosterone, almost 4-foldlowerinpatientswithnonclassic 21OHD 21OHD ( albeit lowerthanpreviouslyshowninpatientswithclassic with nonclassic21OHDthaninunaffectedindividuals, 16OHP4 and 21dFare also significantly higherin patients individuals. In addition to 17OHP4, we found that Steroid biomarkersinN21OHD 32 30 β ). In menstruating girls and women, baseline blood ). Similarly, theratioof(17OHP4 -hydroxylase (CYP11B1).Inunaffectedindividuals, A secondclinicalchallenge withnonclassic21OHD 32 16 ), duetothedualadrenalandgonadalorigin ). Conversely, corticosteronewasonaverage Downloaded fromBioscientifica.com at09/29/202106:56:02PM 33 https://eje.bioscientifica.com ). To date, however, data 183 32 + :1 ). Symptomsof A4)/cortisol was 67 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com group), allwithdisproportionately higher11OHA4thanA4. panels (twofromtheN21OHD groupandonefromthecontrol patients, threeoutlierswereexcluded forthe11OH4andA4 illustrate thecorrelationtrends forthemajorityof androstenedione; 11OHA4,11 T, testosterone;11KT,11-ketotestosterone;A4, nonclassic 21-hydroxylasedeficiency(N21OHD)andcontrols. Correlations betweenkeyandrogensinfemalepatientswith Figure 2 21OHD. Inourstudy, A4andtestosteroneweresimilar have beenmoreinconsistentinadultswithnonclassic children withnonclassic21OHD( testosterone andA4elevationshavebeenreportedin vs adrenalcomponentsofandrogenexcess.Although steroids havebeenunreliableindistinguishingtheovarian in womenwithnonclassic21OHD( assessment, polycysticovarianmorphologyiscommon miscarriages ( hirsutism, acne,irregularmenses,andinfertilityor indistinguishable fromthoseseeninPCOSandinclude 11-ketotestosterone. A4, androstenedione;F,females;11OHA4,11 *three pre-pubertalgirlswereexcludedfromthisgroup,topreservecomparableagesbetweenthetwogroups. 11KT (nmol/L) 11OHT (nmol/L) 11KA4 (nmol/L) 11OHA4 (nmol/L) Testosterone (nmol/L) A4 (nmol/L) BMI (kg/m Age (years) expressed asmedian(interquartilerange). Table 4 Clinical Study Comparison betweenwomenwithnonclassic21-hydroxylasedeficiency(N21OHD)andPCOS.Dataare 2 ) 2 , 3 , 4 ). To furthercomplicatetheclinical β -hydroxyandrostenedione. To F N21OHD* 13.2 (7.6–33.4) 2.7 (1.2–3.8) 1.3 (0.8–2) 1.3 (0.7–1.8) 1.4 (0.9–2.7) 5.4 (3.8–8.3) 27 (23–33) 30 (23–36) A FTurcuandothers β -hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione;11OHT11 34 ), theseandrogens ( 5 n = 22) ). Conventional 31 (27–36) 28 (22–35) 1.0 (0.6–1.3) 0.5 (0.4–0.7) 0.7 (0.6–0.9) 4.4 (3.1–8.6) 2.1 (1–4.2) 5.9 (4–9.9) PCOS ( n = 27) control inpatientswithclassic21OHD( and correlateswellwithclinicalsurrogatesofpoordisease than intheircounterparts.We havepreviouslyshownthat roughly twiceashighinpatientswithnonclassic21OHD to women.Importantly, however, 11-oxyandrogenswere 21OHD, includingwhenthedataanalysiswasrestricted in patientswithnonclassic21OHDandthosewithout cases. Extendedtolarger populations, similarsteroid the needforcosyntropin-stimulation testinginequivocal the addedvalueofmulti-steroid panelscouldcircumvent even individuallywhenmeasured bymassspectrometry, power 17OHP4 and21dFdisplayexcellent discriminatory of nonlcassic21OHDwithasinglebaselinebloodtest.While facilitates the diagnosis quantitation by mass spectrometry component oftestosteroneexcess. female patientswithout21OHDpointstowardanovarian the weaker correlation between 11KT and testosterone in elevations ofadrenalorigininthesepatients.Incontrast, patients withnonclassic21OHD,suggestingtandem a tightpositivecorrelationwithtestosteroneinfemale with sharedclinicalfeatures.Moreover, 11KTdisplayed 21OHD ascomparedtootherdisordersofandrogenexcess disproportionately higher in patients with nonclassic suggest that,overall, the adrenal11-oxyandrogens are isolated hirsutismormenstrualdysfunction.Ourfindings excluded werediagnosedwithPCOSandmostothershad study, halfofthefemalepatientsinwhich21OHDwas with PCOSthanincontrolsofsimilarages( 11-oxyandrogens werealsofoundtobehigherinwomen manifestations of normalandprematureadrenarche ( human beings ( is recognizedasapotentandrogenofadrenaloriginin therapy thaninsex-andage-matchedcontrols( classic 21OHDduringtreatmentwithstandardhormonal 11-oxyandrogens were 3–4foldhigherinpatientswith Steroid biomarkersinN21OHD In summary, wehavefound thataccuratesteroid Ratio ofmediansN21OHD/Control 35 , 36 2.6 2.8 1.9 3.0 0.7 0.9 Downloaded fromBioscientifica.com at09/29/202106:56:02PM ,

37 β ), which mediates clinical -hydroxytestosterone; 11KT, 183 24 :1 ). Interestingly, 38 < < < 22 0.0001 0.0001 0.0054 0.0001 0.24 0.37 0.12 0.73 ). Inour P ). 11KT 68 35 via freeaccess ) European Journal of Endocrinology for theirassistancewithregulatoryprocesses, andallstudyparticipants. LC-MS/MS assays, the University of Michigan adrenal research coordinators with assistance their for O’Day Patrick and Ren Jianwei thank authors The Acknowledgements R JAandDPMcontributedequallytothisstudy. Author contributionstatement the by and T, F Intramural ResearchProgramoftheNationalInstitutesHealth. A to Microgrant Foundation and Hospital Foundation Children’s Disease BC Rare the the by T, F A to and Award/U046500 Science T) the F by Health, A of Institutes (to National Michigan Institute for Clinical and HealthResearch(MICHR) Translational the 1K08DK109116 from A) grants J R by (to R01GM086596 supported was work This Funding Spruce Biosciences. Theotherauthorshavenothingtodisclose. and Biosciences, Neurocrine Therapeutics, Millendo from funds research unrelated received A J R Agreement. Development and Research Therapeutics through the National Institutes of Health Cooperative D P M received unrelated research funds from Diurnal Limited and Millendo Declaration ofinterest in clinicalpractice. biosynthesis, theirclinicalsignificance,andutility the mechanismsresponsiblefor11-oxyandrogen hyperandrogenism inthesewomen,willfurtherelucidate PCOS, alongwithcarefulcorrelationstigmataof biomarkers between women with nonclassic 21OHD and Further studiesfocusingdirectlyonthecomparisonof a heterogeneousspectrumofagesandpathologies. Additionally, patientswithout21OHDcomprised and incompleteclinicalphenotypinginsomepatients. study includeitsmoderatesamplesizewithfewmales other disordersofandrogenexcess.Limitationsour with nonclassic 21OHD as compared to individuals with that 11-oxyandrogensarerelativelyhigherinpatients cosyntropin stimulation ( single baselinesamples,aswaspreviouslyproposedwith nonclassic, carriersof21OHD,andhealthyindividualsin panels could potentially differentiate between classic, OR, oddsratio;17OHP4,17 Corticosterone 21dF 17OHP4 Steroid deficiency andunaffectedindividuals. Table 5 Clinical Study Logistic regressionmodelutilizingthreesteroidstodiscriminatebetweenpatientswithnonclassic21-hydroxylase α -hydroxyprogesterone; 21dF,21-deoxycortisol. 17 , 18 ). Second, we now show A FTurcuandothers Coefficient − 0.43 1.46 0.05 References

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