Anouk Olthof^1,2,3, Marelle J. Bouva^1,3,4, Hedi L. Claahsen – van der Grinten⁵, Dineke Westra⁶, Eugènie Dekkers⁷, Annemieke C. Heijboer^1,2,3,8, Sabine E. Hannema^3,8,9,10, Anita Boelen^1,3,8

1. Endocrine Laboratory, Department of Laboratory Medicine, Amsterdam UMC location University of Amsterdam, the Netherlands
2. Endocrine Laboratory, Department of Laboratory Medicine, Amsterdam UMC location Vrije Universiteit Amsterdam, the Netherlands
3. Amsterdam Gastroenterology, Endocrinology & Metabolism, Amsterdam, the Netherlands
4. Centre for Health Protection, Department of Public Health Genomics & Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
5. Radboud university medical center, Amalia Childrens Hospital, Department of Pediatric Endocrinology, Nijmegen, the Netherlands
6. Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
7. Centre for Population Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
8. Amsterdam Reproduction and Development, Amsterdam, the Netherlands 
9. Department of Pediatric Endocrinology, Emma Children’s Hospital, Amsterdam UMC location University of Amsterdam, Amsterdam, the Netherlands
10. Department of Pediatric Endocrinology, Amsterdam UMC location Vrije Universiteit Amsterdam, Amsterdam, the Netherlands

Background:

Congenital adrenal hyperplasia (CAH) is a group of inherited adrenal steroid synthesis disorders. In the Dutch Newborn Screening (NBS) for CAH, 17-hydroxyprogesterone (17-OHP) is measured in dried blood spots (DBS) with gestational age–adjusted cutoffs followed by 21-deoxycortisol (21-DOCL) as a second-tier test in case the 17-OHP results are inconclusive. Since the implementation of the second-tier in October 2021, a few newborns were referred to the hospital based on the 21-DOCL measurement but were categorized false-positive based on the biochemical confirmation tests. The objective of this study was to further characterize the specificity of the second-tier test by performing DNA analysis of the DBS of these newborns.

Methods:

Mutation analysis of CYP21A2, CYP11B1, POR and HSD3B2 was performed on DNA from the residual DBS from newborns with a false-positive second-tier result.

Results:

Over a period of 5 years, eight false-positive second-tier results were reported (mean 21-DOCL: 7 nmol/L blood), material of six newborns was available. In five out of six samples with a positive 21-DOCL, biallelic pathogenic variants in the CYP21A2 gene were found, related to the non-classical phenotype. Four of them were term newborns with a normal birthweight (mean 3729g, range 3214-4070g), showing a first-tier 17-OHP concentration just on or above the cutoff of 25 nmol/L blood. The other sample had no mutations in any of the genes.

Conclusion:

DNA analysis of the false-positive second-tier DBS revealed in 83% pathogenic variants in the CYP21A2 gene associated with non-classical CAH. To further improve the screening and reduce the chance of detecting non-classical CAH via a positive second-tier, it might be beneficial to investigate adjusting the cutoff value for the inconclusive category of the first-tier 17-OHP measurement.