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Diagnosis of 21-hydroxylase deficient non-classic congenital adrenal hyperplasia and heterozygosity by liquid chromatography and tandem mass spectrometry (#58)

Jennifer Ng ¹ ² , Ee Mun Lim ³ ⁴ , Rui Zhang ³ , John Beilby ⁵ , Gerald Watts ⁶ ⁷ , Bronwyn Stuckey ² ⁴ ⁷

Hollywood Consulting Centre, Nedlands, WA, Australia
The Keogh Institute for Medical Research, Nedlands, WA, Australia
Chemical Pathology, PathWest QEII Laboratory, Nedlands, WA, Australia
Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, WA, Australia
School of Biomedical Sciences, University of Western Australia , Nedlands, WA
Head of Cardiometabolic Service, Royal Perth Hospital, Perth, WA, Australia
Medicine and Pharmacology, University of Western Australia, Perth, WA, Australia

NCCAH is a key differential diagnosis for PCOS. NCCAH is diagnosed via the SST when 17OHP is >30 nmol/L. Increasing use of LC-MS/MS necessitates revision of this immunoassay-derived threshold. Heterozygotes (HTZ) may present for investigation of hyperandrogenic symptoms [1]. As 17OHP is normal in 20-70% of HTZ, diagnosis relies on expensive molecular studies. A biochemical test for HTZ would economise use of molecular studies and support genetic counselling when genotyping is unavailable. 21-deoxycortisol (21-DF) may be useful for HTZ diagnosis[2-4].

We aimed to define LC-MS/MS-specific criteria for NCCAH and HTZ.

From Pathwest QEII laboratory database, we identified genotyped females>15yrs who had undergone an SST_LC-MS/MS from January 2010 to June 2017, and prospectively recruited hyperandrogenic females referred for an SST from June 2017 to August 2021. Steroids were compared among genetically confirmed PCOS, HTZ and NCCAH.

17 OHP_LC-MS/MS results were available for n=81 (53 PCOS, 19 HTZ and 9 NCCAH). 21-DF was also estimated in n=36 (21 PCOS, 9 HTZ and 6 NCCAH). The best single parameter to discriminate HTZ from PCOS was 21-DF_30mins.

Diagnostic thresholds to distinguish HTZ (plus NCCAH) from PCOS
Parameter	Optimal Threshold	sensitivity	specificity	AUC	95% CI	p-value
21-DF_30mins	1.01 nmol/L	100%	85.7%	0.981	0.947-1.01	<0.001
17 OHP_peak	8.05 nmol/L	100%	81.1%	0.950	0.909-0.991	<0.001
(21DF+17OHP)/cortisol_60minsx1000	13.6 (unitless)	100%	88.2%	0.982	0.946-1.018	<0.001
Diagnostic thresholds for NCCAH
Parameter	Optimal Threshold	sensitivity	specificity	AUC	95% CI	p-value
Basal 17 OHP	3.55 nmol/L	93.3%	88.6%	0.979	0.948-1.01	<0.001
17 OHP_peak	20.7 nmol/L	100%	98.6%	1.00	1.00-1.00	<0.001
Basal 21-DF	0.31 nmol/L	100%	96.7%	0.994	0.977-1.01	<0.001
21-DF_peak	13.31 nmol/L	100%	100%	1.00	1.00-1.00	<0.001

Conclusion: ACTH-stimulated 21-DF and 17OHP measured by LC-MS/MS permit excellent discrimination between HTZ and PCOS. Thresholds for NCCAH are lower by LC-MS/MS than those defined by immunoassays.

1. Neocleous, V., et al., Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations. Indian J Endocrinol Metab, 2014. 18(Suppl 1): p. S72-9.
2. Costa-Barbosa, F.A., et al., Superior discriminating value of ACTH-stimulated serum 21-deoxycortisol in identifying heterozygote carriers for 21-hydroxylase deficiency. Clinical Endocrinology, 2010. 73(6): p. 700-706.
3. Costa-Barbosa, F.A., et al., Reassessment of predictive values of ACTH-stimulated serum 21-deoxycortisol and 17-hydroxyprogesterone to identify CYP21A2 heterozygote carriers and nonclassic subjects. Clin Endocrinol (Oxf), 2021.
4. Kulle, A.E., et al., LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers. European Journal of Endocrinology, 2015. 173(4): p. 517-24.

Oral Virtual Presentation (Virtual only) ESA-SRB-ANZBMS 2021

Diagnosis of 21-hydroxylase deficient non-classic congenital adrenal hyperplasia and heterozygosity by liquid chromatography and tandem mass spectrometry (#58)