Can Incorporating Molecular Testing Improve the Accuracy of Newborn Screening for Congenital Adrenal Hyperplasia?

Abstract

Context: Single-tier newborn screening (NBS) for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24 to 48 hours produces a high false-positive rate (FPR). Second-tier steroid testing can reduce the FPR and has been widely implemented.

Objective: We investigated the accuracy of an alternative multitier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the first-tier 17OHP cutoff to minimize missed cases.

Methods: We create a Minnesota-specific CYP21A2 pathogenic variants panel; developed a rapid, high-throughput multiplex, allele-specific-primer-extension assay; and performed a 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based 2-tier protocol and a molecular-based multitier NBS protocol, applied post hoc.

Results: CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. The Centers for Disease Control and Prevention created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68 659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A 3-tier protocol with a lower first-tier steroid cutoff, second-tier 21-variant CYP21A2 panel, and third-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs, and 3 TPs.

Conclusion: Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs.

Keywords: CYP21A2 gene; congenital adrenal hyperplasia; newborn screening; premature infants.

Figure 1.

Long-range amplification-based method (LRAM) to detect A, the CYP21A2 gene; B, 30-kb deletion; and C, large gene conversion alleles. A, The CYP21A1P pseudogene and CYP21A2 gene are part of a 30-kb chromosomal duplication. B, Recombination between CYP21A1P and CYP21A2 deletes a 30-kb section of the repeat forming a fusion of the 5′ end of the pseudogene with the 3′ portion of CYP21A2. C, A unidirectional transfer of pseudogene sequence to CYP21A2 in a large-scale gene conversion results in a fusion of the 5′ end of CYP21A2 with the 3′ end of CYP21A1P.

Figure 2.

Algorithm of molecular-based protocol for newborn screening for congenital adrenal hyperplasia (CAH). A, Molecular protocol—all second-tier CYP21A2 samples with one or more pathogenic variants sent to third-tier sequencing. If 2 or more pathogenic variants are confirmed, the newborn was referred to pediatric endocrinologist for serum 17-hydroxyprogesterone (17OHP) and/or adrenocorticotropin stimulation confirmation testing.

Figure 3.

Algorithm of steroid-based protocol for newborn screening for congenital adrenal hyperplasia (CAH). *17-Hydroxyprogesterone (17OHP) value greater than or equal to 100 ng/mL and gestation age greater than 37 weeks are sent directly to confirmatory testing.