Identification of heterozygotic carriers of 21-hydroxylase deficiency: Sensitivity of ACTH stimulation tests

Abstract

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. To ascertain carrier status, adrenocorticotropin (ACTH) stimulation tests are often used. To determine the sensitivity of ACTH stimulation to detect heterozygotes and to correlate stimulated 17-hydroxyprogesterone responses with molecular genotype, we compared molecular genetic analysis of the 21-hydroxylase (CYP21) gene with 17-hydroxyprogesterone responses at 30 min in 51 individuals. Molecular genotype analysis and ACTH stimulation tests were performed in healthy volunteers (n = 20) and relatives of patients with congenital adrenal hyperplasia (n = 31). Polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, allele-specific oligonucleotide hybridization (ASOH) analysis, and restriction fragment length polymorphism (RFLP) analysis were utilized to screen for 14 CYP21 mutations which account for >90% of the mutations associated with 21-hydroxylase deficiency. Molecular genotype analysis classified 28 individuals as heterozygotic carriers and 23 individuals as normal for all mutations tested. As a group, the heterozygotes had significantly greater stimulated 17-hydroxyprogesterone responses at 10 and 30 min (P < 0.0005). However, on an individual basis, 14/28 (50%) genotyped heterozygotic carriers had stimulated 17-hydroxyprogesterone concentrations, 17-hydroxyprogesterone/cortisol ratios, and 17-hydroxyprogesterone incremental elevations indistinguishable from the genotyped normal individuals. Thus, a normal 17-hydroxyprogesterone response to ACTH stimulation testing does not exclude carrier status for 21-hydroxylase deficiency. Molecular genotype analysis is a more reliable method to determine 21-hydroxylase heterozygotes.

Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status

“…This is in agreement with observations that heterozigosity for CYP21 mutations may result in mild abnormalities of androgen biosynthesis reflected by higher mean free testosterone (13,30) or by higher mean DHEAS concentrations (10). Our results suggest that, in contrast with findings in patients with NC-CAH, among hyperandrogenic women, basal or ACTHstimulated 17-OHP concentrations are not a good indicator of the carrier status for inherited defects of CYP21, as observed also by other investigators (13,31,32) who defined the carrier status by molecular analysis of CYP21 gene, the most accurate method to define carriers for inherited defects of CYP21 (10). Older studies reported that the majority of heterozygotes for 21-hydroxylase deficiency can be detected by measuring the 17-OHP concentration 60 min after ACTH stimulation; however, the diagnosis of 21-hydroxylase deficiency was based on HLA genotyping (33 It remains unclear what was the cause of borderline elevations in ACTH-stimulated 17-OHP concentrations in nine of 12 hyperandrogenic patients (75%), in whom even extensive sequencing of the CYP21 gene and its proximal promoter region revealed no mutations.…”

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

“…At the hormone level, heterozygotes have normal or only slightly elevated basal plasma levels of 17-hydroxyprogesterone (17OHP) (3). Following corticotrophin (ACTH) injection, heterozygotes usually show an increased rise of 17OHP compared with controls, but it is not possible to identify heterozygosity based on stimulated 17OHP alone (11,12,13,14). A different approach to detect heterozygotes is the calculation of substrate-to-product ratios.…”

Congenital Adrenal Hyperplasia

“…33 Whereas an ACTH-stimulated17-OHP concentration less than 400 mg/dl is reassuring that the person is not a carrier, molecular genetic testing may be needed to completely exclude carrier status. 86 …”